β2-adrenergic receptor agonists

ABSTRACT

Disclosed are multibinding compounds which are β2 adrenergic receptor agonists and are useful in the treatment and prevention of respiratory diseases such as asthma, bronchitis. They are also useful in the treatment of nervous system injury and premature labor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/108,945, filed Mar. 28, 2002, now U.S. Pat. No. 6,916,961 which is acontinuation of U.S. application Ser. No. 09/637,899, filed Aug. 14,2000, now U.S. Pat. No. 6,576,793 B1, which is a continuation of U.S.application Ser. No. 09/457,618, filed Dec. 8, 1999, now abandoned, theentire disclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel multibinding compounds (agents) that areβ2 adrenergic receptor agonists, partial agonists and pharmaceuticalcompositions comprising such compounds. Accordingly, the multibindingcompounds and pharmaceutical compositions of this invention are usefulin the treatment and prevention of respiratory diseases such as asthma,chronic obstructive pulmonary disease and chronic bronchitis. They arealso useful in the treatment of nervous system injury and prematurelabor.

REFERENCES

The following publications are cited in this application as superscriptnumbers:

-   -   ¹ Hardman, J. G., et al. “The Pharmacological Basis of        Therapeutics”, McGraw-Hill, New York, (1996)    -   ² Strosberg, A. D. “Structure, Function, and Regulation of        Adrenergic Receptors” Protein Sci. 2, 1198–1209 (1993).    -   ³ Beck-Sickinger, A. G. “Structure Characterization and Binding        Sites of G-Protein-coupled Receptors” DDT, 1, 502–513, (1996).    -   ⁴ Hein, L. & Kobilka, B. K. “Adrenergic Receptor Signal        Transduction and Regulation” Neuropharmacol, 34, 357–366,        (1995).    -   ⁵ Strosberg, A. D. & Pietri-Rouxel, F. “Function, and Regulation        of β3-Adrenoceptor” TiPS, 17, 373–381, (1996).    -   ⁶ Barnes, P. J. “Current Therapies for Asthma” CHEST, 111:        17S–26S, (1997).    -   ⁷ Jack, D. A. “A way of Looking at Agonism and Antagonism:        Lessons from Salbutamol, Salmeterol and other β-Adrenoceptor        Agonists” Br. J. Clin. Pharmac. 31, 501–514, (1991).    -   ⁸ Kissei Pharmaceutical Co. Ltd.        “2-Amino-1-(4-hydroxy-2-methyl-phenyl)propanol derivatives”        JP-10152460 (Publication date Jun. 9, 1998).

All of the above publications are herein incorporated by reference intheir entirety to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein its entirety.

2. State of the Art

A receptor is a biological structure with one or more binding domainsthat reversibly complexes with one or more ligands, where thatcomplexation has biological consequences. Receptors can exist entirelyoutside the cell (extracellular receptors), within the cell membrane(but presenting sections of the receptor to the extracellular milieu andcytosol), or entirely within the cell (intracellular receptors). Theymay also function independently of a cell (e.g., clot formation).Receptors within the cell membrane allow a cell to communicate with thespace outside of its boundaries (i.e., signaling) as well as to functionin the transport of molecules and ions into and out of the cell.

A ligand is a binding partner for a specific receptor or family ofreceptors. A ligand may be the endogenous ligand for the receptor oralternatively may be a synthetic ligand for the receptor such as a drug,a drug candidate or a pharmacological tool.

The super family of seven transmembrane proteins (7-TMs), also calledG-protein coupled receptors (GPCRs), represents one of the mostsignificant classes of membrane bound receptors that communicate changesthat occur outside of the cell's boundaries to its interior, triggeringa cellular response when appropriate. The G-proteins, when activated,affect a wide range of downstream effector systems both positively andnegatively (e.g., ion channels, protein kinase cascades, transcription,transmigration of adhesion proteins, and the like).

Adrenergic receptors (AR) are members of the G-protein coupled receptorsthat are composed of a family of three receptor sub-types: α1 (_(A,B,D))α2 (_(A,B,C)) and β (_(1,2,3)).¹⁻⁵ These receptors are expressed intissues of various systems and organs of mammals and the proportions ofthe α and the β receptors are tissue dependant. For example, tissues ofbronchial smooth muscle express largely β2-AR while those of cutaneousblood vessels contain exclusively α-AR subtypes.

It has been established that the β2-AR sub-type is involved inrespiratory diseases such as such as asthma⁶, chronic bronchitis,nervous system injury, and premature labor⁸. Currently, a number ofdrugs e.g., albuterol, formoterol, isoprenolol, or salmeterol havingβ2-AR agonist activities are being used to treat asthma. However, thesedrugs have limited utility as they are either non-selective therebycausing adverse side effects such as muscle tremor, tachycardia,palpitations, and restlesness⁶, or have short duration of action and/orslow onset time of action.⁷ Accordingly, there is a need forβ2-selective AR agonists that are fast acting and have increased potencyand/or longer duration of action.

The multibinding compounds of the present invention fulfill this need.

SUMMARY OF THE INVENTION

This invention is directed to novel multibinding compounds (agents) thatare agonists or partial agonists of β2 adrenergic receptor and aretherefore useful in the treatment and prevention of respiratory diseasessuch as asthma, chronic obstructive pulmonary disease, and chronicbronchitis. They are also useful in the treatment of nervous systeminjury and premature labor.

Accordingly, in one of its composition aspects, this invention providesa multibinding compound of Formula (I):(L)_(p)(X)_(q)  (I)wherein:

p is an integer of from 2 to 10;

q is an integer of from 1 to 20;

X is a linker; and

L is a ligand wherein:

one of the ligands, L, is a compound of formula (a):

wherein:

Ar¹ and Ar² are independently selected from the group consisting ofaryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclylwherein each of said Ar¹ and Ar² substituent optionally links the ligandto a linker;

R¹ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl, or R¹ is a covalent bond linking the ligand to alinker;

R² is selected from the group consisting of hydrogen, alkyl, aralkyl,acyl, substituted alkyl, cycloalkyl, and substituted cycloalkyl, or R²is a covalent bond linking the ligand to a linker;

W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene or substituted alkylene group is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker; and

the other ligands are independently of each other a compound of formula(b):-Q-Ar³  (b)wherein:

Ar³ is selected from the group consisting of aryl, heteroaryl,cycloalkyl, substituted cycloalkyl, and heterocyclyl;

Q, which links the other ligand to the linker, is selected from thegroup consisting of a covalent bond, alkylene, and substituted alkylenewherein one or more of the carbon atoms in said alkylene and substitutedalkylene is optionally replaced by a substituent selected from the groupconsisting of —NR^(a)— (where R^(a) is hydrogen, alkyl, acyl, or acovalent bond linking the ligand to a linker), —O—, —S(O)_(n)— (where nis an integer of from 0 to 2), —CO—, —PR^(b)— (where R^(b) is alkyl),—P(O)₂—, and —O—P(O)O—; and

individual isomers, mixtures of isomers and pharmaceutically acceptablesalts thereof provided that:

-   (i) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ and Ar³ are aryl, then W and X both are not alkylene oralkylene-O—;

-   (ii) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is aryl orheterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, then thelinker X is not linked to the Ar² group through an oxygen atom;

-   (iii) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ and Ar³ are aryl, W is alkylene, Ar² is aryl or cycloalkyl, Qis a covalent bond, then X is not -alkylene-O—; and

-   (iv) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ is 4-benzyloxy-3-formylamino, R² is aralkyl, W is—CH(CH₃)CH₂—, Ar² and Ar³ are phenyl, Q is a covalent bond, then thelinker X is not linked to the Ar² group through an oxygen atom.

More preferably, each linker, X, in the multibinding compound of Formula(I) independently has the formula:—X^(a)-Z-(Y^(a)-Z)_(m)-X^(a)—wherein

m is an integer of from 0 to 20;

X^(a) at each separate occurrence is selected from the group consistingof —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, C(S),—C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where R is as definedbelow;

Z at each separate occurrence is selected from the group consisting ofalkylene, substituted alkylene, cycloalkylene, substitutedcycloalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—,—NR′—C(X^(a))═N—, —P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′—, —NR′—S(O)_(n)—, —S—S—, and a covalent bond; where n is0, 1 or 2; R, R′ and R″ at each separate occurrence are selected fromthe group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic, and X^(a) is as defined above.

Preferably, q is less than p in the multibinding compounds of thisinvention.

In still another of its composition aspects, this invention provides apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount of a multibinding compound of Formula(I):(L)_(p)(X)_(q)  (I)wherein:

p is an integer of from 2 to 10;

q is an integer of from 1 to 20;

X is a linker; and

L is a ligand wherein:

one of the ligands, L, is a compound of formula (a):

wherein:

Ar¹ and Ar² are independently selected from the group consisting ofaryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclylwherein each of said Ar¹ and Ar² substituent optionally links the ligandto a linker;

R¹ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl, or R¹ is a covalent bond linking the ligand to alinker;

R² is selected from the group consisting of hydrogen, alkyl, aralkyl,acyl, substituted alkyl, cycloalkyl, and substituted cycloalkyl, or R²is a covalent bond linking the ligand to a linker;

W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene and substituted alkylene is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker; and

-   -   the other ligands are independently of each other a compound of        formula (b):        -Q-Ar³  (b)        wherein:

Ar³ is selected from the group consisting of aryl, heteroaryl,cycloalkyl, substituted cycloalkyl, and heterocyclyl;

Q, which links the other ligand to the linker, is selected from thegroup consisting of a covalent bond, alkylene, or a substituted alkylenegroup wherein one or more of the carbon atoms in said alkylene orsubstituted alkylene group is optionally replaced by a substituentselected from the group consisting of —NR^(a)— (where R^(a) is hydrogen,alkyl, acyl, or a covalent bond linking the ligand to a linker), —O—,—S(O)_(n)— (where n is an integer of from 0 to 2), —CO—, —PR^(b)— (whereR^(b) is alkyl), —P(O)₂—, and —O—P(O)O—; and

individual isomers, mixtures of isomers and pharmaceutically acceptablesalts thereof provided that:

-   (i) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ and Ar³ are aryl, then W and X both are not alkylene oralkylene-O—;

-   (ii) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is aryl orheterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, then thelinker X is not linked to the Ar² group through an oxygen atom;

-   (iii) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹, Ar², Ar³, R¹, R² are as defined above, W is alkylene, and Qis a covalent bond, then X is not -alkylene-O—; and

-   (iv) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ is 4-benzyloxy-3-formylamino, R² is aralkyl, W is—CH(CH₃)CH₂—, Ar² and Ar³ are phenyl, Q is a covalent bond, then thelinker X is not linked to the Ar² group through an oxygen atom.

More preferably, each linker, X, in the multibinding compound of Formula(I) independently has the formula:—X^(a)-Z-(Y^(a)-Z)_(m)-X^(a)—wherein

m is an integer of from 0 to 20;

X^(a) at each separate occurrence is selected from the group consistingof —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, C(S),—C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where R is as definedbelow;

Z at each separate occurrence is selected from the group consisting ofalkylene, substituted alkylene, cycloalkylene, substitutedcycloalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—,—NR′—C(X^(a))═N—, —P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′—, —NR′—S(O)_(n)—, —S—S—, and a covalent bond; where n is0, 1 or 2; R, R′ and R″ at each separate occurrence are selected fromthe group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic, and X^(a) is as defined above.

In still another aspect, this invention provides a method of treatingdiseases mediated by a β2 adrenergic receptor in a mammal, said methodcomprising administering to said mammal a therapeutically effectiveamount of a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a multibinding compound of Formula (I):(L)_(p)(X)_(q)  (I)wherein:

p is an integer of from 2 to 10;

q is an integer of from 1 to 20;

X is a linker; and

L is a ligand wherein:

one of the ligands, L, is a compound of formula (a):

wherein:

Ar¹ and Ar² are independently selected from the group consisting ofaryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclylwherein each of said Ar¹ and Ar² substituent optionally links the ligandto a linker;

R¹ is selected from the group consisting of hydrogen, alkyl, andsubstituted alkyl, or R¹ is a covalent bond linking the ligand to alinker;

R² is selected from the group consisting of hydrogen, alkyl, aralkyl,acyl, substituted alkyl, cycloalkyl, and substituted cycloalkyl, or R²is a covalent bond linking the ligand to a linker;

W is a covalent bond linking the —NR²— group to Ar², alkylene orsubstituted alkylene wherein one or more of the carbon atoms in saidalkylene and substituted alkylene group is optionally replaced by asubstituent selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, acyl, or a covalent bond linking the ligand to alinker), —O—, —S(O)_(n) (where n is an integer of from 0 to 2), —CO—,—PR^(b)— (where R^(b) is alkyl), —P(O)₂—, and —O—P(O)O— and furtherwherein said alkylene or substituted alkylene group optionally links theligand to a linker provided that at least one of Ar¹, Ar², R¹, R², or Wlinks the ligand to a linker, and

the other ligands are independently of each other a compound of formula(b):-Q-Ar³  (b)wherein:

Ar³ is selected from the group consisting of aryl, heteroaryl,cycloalkyl, substituted cycloalkyl, and heterocyclyl;

Q, which links the other ligand to the linker, is selected from thegroup consisting of a covalent bond, alkylene, and substituted alkylenewherein one or more of the carbon atoms in said alkylene and substitutedalkylene group is optionally replaced by a substituent selected from thegroup consisting of —NR^(a)— (where R^(a) is hydrogen, alkyl, acyl, or acovalent bond linking the ligand to a linker), —O—, —S(O)_(n)— (where nis an integer of from 0 to 2), —CO—, —PR^(b)— (where R^(b) is alkyl),—P(O)₂—, and —O—P(O)O—; and

individual isomers, mixtures of isomers and pharmaceutically acceptablesalts thereof provided that:

-   (i) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ and Ar³ are aryl, then W and X both are not alkylene oralkylene-O—;

-   (ii) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ is 4-hydroxy-2-methylphenyl, Ar² is aryl, Ar³ is aryl orheterocyclyl, W is ethylene, Q is a covalent bond, R¹ is alkyl, then thelinker-X is not linked to the Ar² group through an oxygen atom;

-   (iii) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹, Ar², Ar³, R¹, R² are as defined above, W is alkylene, and Qis a covalent bond, then X is not -alkylene-O—; and

-   (iv) when the multibinding compound of Formula (I) is a compound of    formula:

where Ar¹ is 4-benzyloxy-3-formylamino, R² is aralkyl, W is—CH(CH₃)CH₂—, Ar² and Ar³ are phenyl, Q is a covalent bond, then thelinker X is not linked to the Ar² group through an oxygen atom.

More preferably, each linker, X, in the multibinding compound of Formula(I) independently has the formula:—X^(a)-Z-(Y^(a)-Z)_(m)-X^(a)—wherein

m is an integer of from 0 to 20;

X^(a) at each separate occurrence is selected from the group consistingof —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, C(S),—C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where R is as definedbelow;

Z at each separate occurrence is selected from the group consisting ofalkylene, substituted alkylene, cycloalkylene, substitutedcycloalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X^(a))—NR′—,—NR′—C(X)═N—, —P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′—, —NR′—S(O)R —, —S—S—, and a covalent bond; where n is 0,1 or 2; R, R′ and R″ at each separate occurrence are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic, and X^(a) is as defined above.

Preferably p is less than q.

In still another aspect, this invention is directed to general syntheticmethods for generating large libraries of diverse multimeric compoundswhich multimeric compounds are candidates for possessing multibindingproperties for β2 adrenergic receptor. The diverse multimeric compoundlibraries provided by this invention are synthesized by combining alinker or linkers with a ligand or ligands to provide for a library ofmultimeric compounds wherein the linker and ligand each havecomplementary functional groups permitting covalent linkage. The libraryof linkers is preferably selected to have diverse properties such asvalency, linker length, linker geometry and rigidity, hydrophilicity orhydrophobicity, amphiphilicity, acidity, basicity and polarization. Thelibrary of ligands is preferably selected to have diverse attachmentpoints on the same ligand, different functional groups at the same siteof otherwise the same ligand, and the like.

This invention is also directed to libraries of diverse multimericcompounds which multimeric compounds are candidates for possessingmultibinding properties for β2 adrenergic receptor. These libraries areprepared via the methods described above and permit the rapid andefficient evaluation of what molecular constraints impart multibindingproperties to a ligand or a class of ligands targeting a receptor.

Accordingly, in one of its method aspects, this invention is directed toa method for identifying multimeric ligand compounds possessingmultibinding properties for β2 adrenergic receptor which methodcomprises:

(a) identifying a ligand or a mixture of ligands wherein each ligandcontains at least one reactive functionality;

(b) identifying a library of linkers wherein each linker in said librarycomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand;

(c) preparing a multimeric ligand compound library by combining at leasttwo stoichiometric equivalents of the ligand or mixture of ligandsidentified in (a) with the library of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands;and

(d) assaying the multimeric ligand compounds produced in (c) above toidentify multimeric ligand compounds possessing multibinding propertiesfor β2 adrenergic receptor.

In another of its method aspects, this invention is directed to a methodfor identifying multimeric ligand compounds possessing multibindingproperties for β2 adrenergic receptor which method comprises:

(a) identifying a library of ligands wherein each ligand contains atleast one reactive functionality;

(b) identifying a linker or mixture of linkers wherein each linkercomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand;

(c) preparing a multimeric ligand compound library by combining at leasttwo stoichiometric equivalents of the library of ligands identified in(a) with the linker or mixture of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands;and

(d) assaying the multimeric ligand compounds produced in (c) above toidentify multimeric ligand compounds possessing multibinding propertiesfor β2 adrenergic receptor.

The preparation of the multimeric ligand compound library is achieved byeither the sequential or concurrent combination of the two or morestoichiometric equivalents of the ligands identified in (a) with thelinkers identified in (b). Sequential addition is preferred when amixture of different ligands is employed to ensure heterodimeric ormultimeric compounds are prepared. Concurrent addition of the ligandsoccurs when at least a portion of the multimer comounds prepared arehomomultimeric compounds.

The assay protocols recited in (d) can be conducted on the multimericligand compound library produced in (c) above, or preferably, eachmember of the library is isolated by preparative liquid chromatographymass spectrometry (LCMS).

In one of its composition aspects, this invention is directed to alibrary of multimeric ligand compounds which may possess multivalentproperties for β2 adrenergic receptor which library is prepared by themethod comprising:

(a) identifying a ligand or a mixture of ligands wherein each ligandcontains at least one reactive functionality;

(b) identifying a library of linkers wherein each linker in said librarycomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand; and

(c) preparing a multimeric ligand compound library by combining at leasttwo stoichiometric equivalents of the ligand or mixture of ligandsidentified in (a) with the library of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands.

In another of its composition aspects, this invention is directed to alibrary of multimeric ligand compounds which may possess multivalentproperties for β2 adrenergic receptor which library is prepared by themethod comprising:

(a) identifying a library of ligands wherein each ligand contains atleast one reactive functionality;

(b) identifying a linker or mixture of linkers wherein each linkercomprises at least two functional groups having complementary reactivityto at least one of the reactive functional groups of the ligand; and

(c) preparing a multimeric ligand compound library by combining at leasttwo stoichiometric equivalents of the library of ligands identified in(a) with the linker or mixture of linkers identified in (b) underconditions wherein the complementary functional groups react to form acovalent linkage between said linker and at least two of said ligands.

In a preferred embodiment, the library of linkers employed in either themethods or the library aspects of this invention is selected from thegroup comprising flexible linkers, rigid linkers, hydrophobic linkers,hydrophilic linkers, linkers of different geometry, acidic linkers,basic linkers, linkers of different polarization and amphiphiliclinkers. For example, in one embodiment, each of the linkers in thelinker library may comprise linkers of different chain length and/orhaving different complementary reactive groups. Such linker lengths canpreferably range from about 2 to 100 Å.

In another preferred embodiment, the ligand or mixture of ligands isselected to have reactive functionality at different sites on saidligands in order to provide for a range of orientations of said ligandon said multimeric ligand compounds. Such reactive functionalityincludes, by way of example, carboxylic acids, carboxylic acid halides,carboxyl esters, amines, halides, isocyanates, vinyl unsaturation,ketones, aldehydes, thiols, alcohols, anhydrides, and precursorsthereof. It is understood, of course, that the reactive functionality onthe ligand is selected to be complementary to at least one of thereactive groups on the linker so that a covalent linkage can be formedbetween the linker and the ligand.

In other embodiments, the multimeric ligand compound is homomeric (i.e.,each of the ligands is the same, although it may be attached atdifferent points) or heterodimeric (i.e., at least one of the ligands isdifferent from the other ligands).

In addition to the combinatorial methods described herein, thisinvention provides for an interative process for rationally evaluatingwhat molecular constraints impart multibinding properties to a class ofmultimeric compounds or ligands targeting a receptor. Specifically, thismethod aspect is directed to a method for identifying multimeric ligandcompounds possessing multibinding properties for β2 adrenergic receptorwhich method comprises:

(a) preparing a first collection or iteration of multimeric compoundswhich is prepared by contacting at least two stoichiometric equivalentsof the ligand or mixture of ligands which target a receptor with alinker or mixture of linkers wherein said ligand or mixture of ligandscomprises at least one reactive functionality and said linker or mixtureof linkers comprises at least two functional groups having complementaryreactivity to at least one of the reactive functional groups of theligand wherein said contacting is conducted under conditions wherein thecomplementary functional groups react to form a covalent linkage betweensaid linker and at least two of said ligands;

(b) assaying said first collection or iteration of multimeric compoundsto assess which if any of said multimeric compounds possess multibindingproperties for β2 adrenergic receptor;

(c) repeating the process of (a) and (b) above until at least onemultimeric compound is found to possess multibinding properties for β2adrenergic receptor;

(d) evaluating what molecular constraints imparted multibindingproperties for β2 adrenergic receptor to the multimeric compound orcompounds found in the first iteration recited in (a)–(c) above;

(e) creating a second collection or iteration of multimeric compoundswhich elaborates upon the particular molecular constraints impartingmultibinding properties to the multimeric compound or compounds found insaid first iteration;

(f) evaluating what molecular constraints imparted enhanced multibindingproperties to the multimeric compound or compounds found in the secondcollection or iteration recited in (e) above;

(g) optionally repeating steps (e) and (f) to further elaborate uponsaid molecular constraints.

Preferably, steps (e) and (f) are repeated at least two times, morepreferably at from 2–50 times, even more preferably from 3 to 50 times,and still more preferably at least 5–50 times.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates examples of multibinding compounds comprising 2ligands attached in different formats to a linker.

FIG. 2 illustrates examples of multibinding compounds comprising 3ligands attached in different formats to a linker.

FIG. 3 illustrates examples of multibinding compounds comprising 4ligands attached in different formats to a linker.

FIG. 4 illustrates examples of multibinding compounds comprising >4ligands attached in different formats to a linker.

FIGS. 5–15 illustrate synthesis of compounds of Formula (I).

DETAILED DESCRIPTION OF THE INVENTION Definitions

This invention is directed to multibinding compounds which are β2adrenergic receptor agonists pharmaceutical compositions containing suchcompounds and methods for treating diseases mediated by β2 adrenergicreceptor in mammals. When discussing such compounds, compositions ormethods, the following terms have the following meanings unlessotherwise indicated. Any undefined terms have their art recognizedmeanings.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 40 carbon atoms,more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6carbon atoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl,and the like.

The term “substituted alkyl” refers to an alkyl group as defined above,having from 1 to 5 substituents, and preferably 1 to 3 substituents,selected from the group consisting of alkoxy, substituted alkoxy,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. This term is exemplified by groups such ashydroxymethyl, hydroxyethyl, hydroxypropyl, 2-aminoethyl, 3-aminopropyl,2-methylaminoethyl, 3-dimethylaminopropyl, 2-sulfonamidoethyl,2-carboxyethyl, and the like.

The term “alkylene” refers to a diradical of a branched or unbranchedsaturated hydrocarbon chain, preferably having from 1 to 40 carbonatoms, more preferably 1 to 10 carbon atoms and even more preferably 1to 6 carbon atoms. This term is exemplified by groups such as methylene(—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers (e.g., —CH₂CH₂CH₂—and —CH(CH₃)CH₂—) and the like.

The term “substituted alkylene” refers to an alkylene group, as definedabove, having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. Additionally, such substituted alkylene groupsinclude those where 2 substituents on the alkylene group are fused toform one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fusedto the alkylene group. Preferably such fused groups contain from 1 to 3fused ring structures.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein. Such alkaryl groups are exemplified by benzyl,phenethyl and the like.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl and-substituted alkylene-heteroaryl where alkylene, substituted alkyleneand heteroaryl are defined herein. Such heteroaralkyl groups areexemplified by pyridin-3-lmethyl, pyridin-3-ylmethyloxy, and the like.

The term “alkoxy” refers to the groups alkyl-O—, alkenyl-O—,cycloalkyl-O—, cycloalkenyl-O—, and alkynyl-O—, where alkyl, alkenyl,cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. Preferredalkoxy groups are alkyl-O— and include, by way of example, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 40 carbonatoms, more preferably 2 to 10 carbon atoms and even more preferably 2to 6 carbon atoms and having at least 1 and preferably from 1–6 sites ofvinyl unsaturation. Preferred alkenyl groups include ethenyl (—CH═CH),n-propenyl (—CH₂CH═CH₂), iso-propenyl (—C(CH₃)═CH), and the like.

The term “substituted alkenyl” refers to an alkenyl group as definedabove having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “alkenylene” refers to a diradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 40 carbonatoms, more preferably 2 to 10 carbon atoms and even more preferably 2to 6 carbon atoms and having at least 1 and preferably from 1–6 sites ofvinyl unsaturation. This term is exemplified by groups such asethenylene (—CH═CH—), the propenylene isomers (e.g., —CH₂CH═CH—,—C(CH₃)═CH—, and the like.

The term “substituted alkenylene” refers to an alkenylene group asdefined above having from 1 to 5 substituents, and preferably from 1 to3 substituents, selected from the group consisting of alkoxy,substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substitutedamino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen,hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Additionally,such substituted alkenylene groups include those where 2 substituents onthe alkenylene group are fused to form one or more cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,heterocyclic or heteroaryl groups fused to the alkenylene group.

The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbonpreferably having from 2 to 40 carbon atoms, more preferably 2 to 20carbon atoms and even more preferably 2 to 6 carbon atoms and having atleast 1 and preferably from 1–6 sites of acetylene (triple bond)unsaturation. Preferred alkynyl groups include ethynyl (—C≡CH),propargyl (—CH₂C≡CH) and the like.

The term “substituted alkynyl” refers to an alkynyl group as definedabove having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,and —SO₂-heteroaryl.

The term “alkynylene” refers to a diradical of an unsaturatedhydrocarbon preferably having from 2 to 40 carbon atoms, more preferably2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms andhaving at least 1 and preferably from 1–6 sites of acetylene (triplebond) unsaturation. Preferred alkynylene groups include ethynylene(—C≡C—), propargylene (—CH₂C≡C—) and the like.

The term “substituted alkynylene” refers to an alkynylene group asdefined above having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl

The term “acyl” refers to the groups HC(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, cycloalkyl-C(O)—,substituted cycloalkyl-C(O)—, cycloalkenyl-C(O)—, substitutedcycloalkenyl-C(O)—, aryl-C(O)—, heteroaryl-C(O)— and heterocyclic-C(O)—where alkyl, substituted alkyl, alkenyl, substituted alkenyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acylamino” or “aminocarbonyl” refers to the group —C(O)NRRwhere each R is independently hydrogen, alkyl, substituted alkyl, aryl,heteroaryl, heterocyclic or where both R groups are joined to form aheterocyclic group (e.g., morpholino) wherein alkyl, substituted alkyl,aryl, heteroaryl and heterocyclic are as defined herein.

The term “sulfonylamino” refers to the group —NRSO₂R^(a) where R ishydrogen, alkyl, substituted alkyl, aralkyl, or heteroaralkyl, and R^(a)is alkyl, substituted alkyl, amino, or substituted amino wherein alkyl,substituted alkyl, aralkyl, heteroaralkyl and substituted amino are asdefined herein.

The term “aminoacyl” refers to the group —NRC(O)R where each R isindependently hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, amino, substituted amino, aryl, heteroaryl, or heterocyclicwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl,heteroaryl and heterocyclic are as defined herein.

The term “aminoacyloxy” or “alkoxycarbonylamino” refers to the group—NRC(O)OR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclic-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclic are as defined herein.

The term “aryl” refers to an unsaturated aromatic carbocyclic group offrom 6 to 20 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed (fused) rings (e.g., naphthyl or anthryl). The arylgroup may optionally be fused to a heterocyclic or cycloalkyl group.Preferred aryls include phenyl, naphthyl and the like. Unless otherwiseconstrained by the definition for the aryl substituent, such aryl groupscan optionally be substituted with from 1 to 5 substituents, preferably1 to 3 substituents, selected from the group consisting of acyloxy,hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, substituted alkyl, substituted alkoxy, substitutedalkenyl, substituted alkynyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino,sulfonylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl,cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substitutedthioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl, —SO-substitutedalkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl,—SO₂-aryl, —SO₂-heteroaryl and trihalomethyl. Preferred arylsubstituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl,and thioalkoxy.

The term “aryloxy” refers to the group aryl-O— wherein the aryl group isas defined above including optionally substituted aryl groups as alsodefined above.

The term “arylene” refers to the diradical derived from aryl (includingsubstituted aryl) as defined above and is exemplified by 1,2-phenylene,1,3-phenylene, 1,4-phenylene, 1,2-naphthylene and the like.

The term “amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, acyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl and heterocyclic provided thatboth R's are not hydrogen.

The term “carboxyalkyl” or “alkoxycarbonyl” refers to the groups“—C(O)O-alkyl”, “—C(O)O-substituted alkyl”, “—C(O)O-cycloalkyl”,“—C(O)O-substituted cycloalkyl”, “—C(O)O-alkenyl”, “—C(O)O-substitutedalkenyl”, “—C(O)O-alkynyl” and “—C(O)O-substituted alkynyl” where alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, alkynyl and substituted alkynyl are as definedherein.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20carbon atoms having a single cyclic ring or multiple condensed rings,said cycloalkyl group may optionally be fused to an aryl or heteroarylgroup. Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, and preferably 1 to 3 substituents, selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 20carbon atoms having a single cyclic ring and at least one point ofinternal unsaturation. Examples of suitable cycloalkenyl groups include,for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and thelike.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, and preferably 1 to 3 substituents, selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbonatoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfurwithin at least one ring (if there is more than one ring). Theheteroaryl ring may optionally be fused to a cycloalkyl or heterocyclylring. Unless otherwise constrained by the definition for the heteroarylsubstituent, such heteroaryl groups can be optionally substituted with 1to 5 substituents, preferably 1 to 3 substituents, selected from thegroup consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.Preferred heteroaryl substituents include alkyl, alkoxy, halo, cyano,nitro, trihalomethyl, and thioalkoxy. Such heteroaryl groups can have asingle ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g.,indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl,pyrrolyl and furyl.

The term “heteroaryloxy” refers to the group heteroaryl-O—.

The term “heteroarylene” refers to the diradical group derived fromheteroaryl (including substituted heteroaryl), as defined above, and isexemplified by the groups 2,6-pyridylene, 2,4-pyridinylene,1,2-quinolinylene, 1,8-quinolinylene, 1,4-benzofuranylene,2,5-pyridnylene, 2,5-indolenyl, and the like.

The term “cycloalkylene” refers to the diradical group derived fromcycloalkyl, as defined above, and is exemplified by the groups1,6-cyclohexylene, 1,3-cyclopentylene, and the like.

The term “substituted cycloalkylene” refers to the diradical groupderived from substituted cycloalkyl, as defined above.

The term “cycloalkenylene” refers to the diradical group derived fromcycloalkyl, as defined above.

The term “substituted cycloalkenylene” refers to the diradical groupderived from substituted cycloalkenyl, as defined above.

The term “heterocycle” or “heterocyclyl” refers to a monoradicalsaturated unsaturated group having a single ring or multiple condensedrings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms,preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within the ring and further wherein one, two,or three of the ring carbon atoms may optionally be replaced with acarbonyl group (i.e., a keto group). Unless otherwise constrained by thedefinition for the heterocyclic substituent, such heterocyclic groupscan be optionally substituted with 1 to 5, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. Such heterocyclic groups can have a single ring ormultiple condensed rings. Preferred heterocyclics include morpholino,piperidinyl, and the like.

Examples of heteroaryls and heterocycles include, but are not limitedto, pyrrole, thiophene, furan, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,pyrrolidine, piperidine, piperazine, indoline, morpholine,tetrahydrofuranyl, tetrahydrothiophene, and the like as well asN-alkoxy-nitrogen containing heterocycles.

The term “heterocyclooxy” refers to the group heterocyclic-O—.

The term “thioheterocyclooxy” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein, and is exemplified by the groups2,6-morpholino, 2,5-morpholino and the like.

The term “oxyacylamino” or “aminocarbonyloxy” refers to the group—OC(O)NRR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “spiro-attached cycloalkyl group” refers to a cycloalkyl groupjoined to another ring via one carbon atom common to both rings.

The term “thiol” refers to the group —SH.

The term “thioalkoxy” or “alkylthio” refers to the group —S-alkyl.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined above including optionally substituted aryl groupsalso defined above.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined above including optionallysubstituted aryl groups as also defined above.

As to any of the above groups which contain one or more substituents, itis understood, of course, that such groups do not contain anysubstitution or substitution patterns which are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the multibindingcompounds of this invention and which are not biologically or otherwiseundesirable. In many cases, the multibinding compounds of this inventionare capable of forming acid and/or base salts by virtue of the presenceof amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically acceptable acid addition salts, may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, naphthoic acid, 2-hydroxynaphthoic acid, and the like.

The term “pharmaceutically-acceptable cation” refers to the cation of apharmaceutically-acceptable salt.

The term “library” refers to at least 3, preferably from 10² to 10⁹ andmore preferably from 10² to 10⁴ multimeric compounds. Preferably, thesecompounds are prepared as a multiplicity of compounds in a singlesolution or reaction mixture which permits facile synthesis thereof. Inone embodiment, the library of multimeric compounds can be directlyassayed for multibinding properties. In another embodiment, each memberof the library of multimeric compounds is first isolated and,optionally, characterized. This member is then assayed for multibindingproperties.

The term “collection” refers to a set of multimeric compounds which areprepared either sequentially or concurrently (e.g., combinatorially).The collection comprises at least 2 members; preferably from 2 to 10⁹members and still more preferably from 10 to 10⁴ members.

The term “multimeric compound” refers to compounds comprising from 2 to10 ligands covalently connected through at least one linker whichcompounds may or may not possess multibinding properties (as definedherein).

The term “pseudohalide” refers to functional groups which react indisplacement reactions in a manner similar to a halogen. Such functionalgroups include, by way of example, mesyl, tosyl, azido and cyano groups.

The term “protecting group” or “blocking group” refers to any groupwhich when bound to one or more hydroxyl, thiol, amino or carboxylgroups of the compounds (including intermediates thereof) preventsreactions from occurring at these groups and which protecting group canbe removed by conventional chemical or enzymatic steps to reestablishthe hydroxyl, thiol, amino or carboxyl group (See., T. W. Greene and P.G. H. Wuts, “Protective Groups in Organic Synthesis”, 2^(nd) Ed.). Theparticular removable blocking group employed is not critical andpreferred removable hydroxyl blocking groups include conventionalsubstituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl,benzylidine, phenacyl, t-butyl-diphenylsilyl and any other group thatcan be introduced chemically onto a hydroxyl functionality and laterselectively removed either by chemical or enzymatic methods in mildconditions compatible with the nature of the product. Preferredremovable thiol blocking groups include disulfide groups, acyl groups,benzyl groups, and the like.

Preferred removable amino blocking groups include conventionalsubstituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ),fluorenylmethoxy-carbonyl (FMOC), allyloxycarbonyl (ALOC), and the likewhich can be removed by conventional conditions compatible with thenature of the product.

Preferred carboxyl protecting groups include esters such as methyl,ethyl, propyl, t-butyl etc. which can be removed by mild conditionscompatible with the nature of the product.

The term “optional” or “optionally” means that the subsequentlydescribed event, circumstance or substituent may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

The term “ligand” or “ligands” as used herein denotes a compound that isa binding partner for a β2 adrenergic receptor and is bound thereto bycomplementarity. Preferred ligands are those that are either β2adrenergic receptor agonists, partial agonists, or antagonists. Thespecific region or regions of the ligand that is (are) recognized by thereceptor is designated as the “ligand domain”. A ligand may be eithercapable of binding to the receptor by itself, or may require thepresence of one or more non-ligand components for binding (e.g., Ca⁺²,Mg⁺² or a water molecule is required for the binding of a ligand tovarious ligand binding sites). Examples of ligands useful in thisinvention are described herein. Those skilled in the art will appreciatethat portions of the ligand structure that are not essential forspecific molecular recognition and binding activity may be variedsubstantially, replaced or substituted with unrelated structures (forexample, with ancillary groups as defined below) and, in some cases,omitted entirely without affecting the binding interaction. The primaryrequirement for a ligand is that it has a ligand domain as definedabove. It is understood that the term ligand is not intended to belimited to compounds known to be useful in binding to β2 adrenergicreceptor (e.g., known drugs). Those skilled in the art will understandthat the term ligand can equally apply to a molecule that is notnormally associated with β2 adrenergic receptor binding properties. Inaddition, it should be noted that ligands that exhibit marginal activityor lack useful activity as monomers can be highly active as multivalentcompounds because of the benefits conferred by multivalency.

The term “ligand” or “ligands” as used herein is intended to include theracemic forms of the ligands as well as individual enantiomers anddiasteromers and non-racemic mixtures thereof.

The term “multibinding compound or agent” refers to a compound that iscapable of multivalency, as defined below, and which has 2–10 ligandscovalently bound to one or more linkers. In all cases, each ligand andlinker in the multibinding compound is independently selected such thatthe multibinding compound includes both symmetric compounds (i.e., whereeach ligand as well as each linker is identical) and asymmetriccompounds (i.e., where at least one of the ligands is different from theother ligand(s) and/or at least one linker is different from the otherlinker(s)). Multibinding compounds provide a biological and/ortherapeutic effect greater than the aggregate of unlinked ligandsequivalent thereto which are made available for binding. That is to saythat the biological and/or therapeutic effect of the ligands attached tothe multibinding compound is greater than that achieved by the sameamount of unlinked ligands made available for binding to the ligandbinding sites (receptors). The phrase “increased biological ortherapeutic effect” includes, for example: increased affinity, increasedselectivity for target, increased specificity for target, increasedpotency, increased efficacy, decreased toxicity, improved duration ofactivity or action, increased ability to kill cells such as fungalpathogens, cancer cells, etc., decreased side effects, increasedtherapeutic index, improved bioavailibity, improved pharmacokinetics,improved activity spectrum, and the like. The multibinding compounds ofthis invention will exhibit at least one and preferably more than one ofthe above-mentioned affects.

The term “univalency” as used herein refers to a single bindinginteraction between one ligand as defined herein with one ligand bindingsite as defined herein. It should be noted that a compound havingmultiple copies of a ligand (or ligands) exhibit univalency when onlyone ligand is interacting with a ligand binding site. Examples ofunivalent interactions are depicted below.

The term “multivalency” as used herein refers to the concurrent bindingof from 2 to 10 linked ligands (which may be the same or different) andtwo or more corresponding receptors (ligand binding sites) which may bethe same or different.

For example, two ligands connected through a linker that bindconcurrently to two ligand binding sites would be considered asbivalency; three ligands thus connected would be an example oftrivalency. An example of trivalent binding, illustrating a multibindingcompound bearing three ligands versus a monovalent binding interaction,is shown below:

It should be understood that not all compounds that contain multiplecopies of a ligand attached to a linker or to linkers necessarilyexhibit the phenomena of multivalency, i.e., that the biological and/ortherapeutic effect of the multibinding agent is greater than the sum ofthe aggregate of unlinked ligands made available for binding to theligand binding site (receptor). For multivalency to occur, the ligandsthat are connected by a linker or linkers have to be presented to theirligand binding sites by the linker(s) in a specific manner in order tobring about the desired ligand-orienting result, and thus produce amultibinding event.

Furthermore, the multibinding compound of the present invention can becomposed of ligands that are all β2 adrenergic receptor agonists,partial agonists, or it can be composed of ligands that are selectedfrom β2 adrenergic receptor agonists and antagonists provided that themultibinding compounds exhibits an overall β2 adrenergic receptoragonistic or partial agonistic activity. A multibinding compound thatexhibits partial agonist activity at adrenoceptors may provideadvantages over a compound that exhibits full agonism. Partial agonismmay result in a reduction of the rate of receptor desensitization,receptor recycling, or receptor expression in mammalian tissue. This mayresult in increased therapeutic benefits from using such an agonistversus a compound which behaves as a full agonist for the chronictreatment of pathological conditions or diseases. A multibindingcompound may also, or separately, act as a tissue-selective partialagonist. For example, a multibinding compound with β2 adrenoceptoragonist activity may exhibit a full maximal response in relaxing smoothmuscle cells in bronchial tissue but exhibit a partial maximal responseto adrenoceptor agonism in cardiac tissue. Thus, a multibinding compoundthat is a tissue-selective partial agonist may provide a lower incidenceof undesired side effects such as positive chronotropism and increasesin cardiac output.

The term “potency” refers to the minimum concentration at which a ligandis able to achieve a desirable biological or therapeutic effect. Thepotency of a ligand is typically proportional to its affinity for itsligand binding site. In some cases, the potency may be non-linearlycorrelated with its affinity. In comparing the potency of two drugs,e.g., a multibinding agent and the aggregate of its unlinked ligand, thedose-response curve of each is determined under identical testconditions (e.g., in an in vitro or in vivo assay, in an appropriateanimal model such a human patient). The finding that the multibindingagent produces an equivalent biological or therapeutic effect at a lowerconcentration than the aggregate unlinked ligand is indicative ofenhanced potency.

The term “selectivity” or “specificity” is a measure of the bindingpreferences of a ligand for different ligand binding sites (receptors).The selectivity of a ligand with respect to its target ligand bindingsite relative to another ligand binding site is given by the ratio ofthe respective values of K_(d) (i.e., the dissociation constants foreach ligand-receptor complex) or, in cases where a biological effect isobserved below the K_(d), the ratio of the respective EC₅₀'s (i.e., theconcentrations that produce 50% of the maximum response for the ligandinteracting with the two distinct ligand binding sites (receptors)).

The term “ligand binding site” denotes the site on the β-adrenergicreceptor that recognizes a ligand domain and provides a binding partnerfor the ligand. The ligand binding site may be defined by monomeric ormultimeric structures. This interaction may be capable of producing aunique biological effect, for example, agonism, antagonism, andmodulatory effects or it may maintain an ongoing biological event, andthe like.

It should be recognized that the ligand binding sites of the receptorthat participate in biological multivalent binding interactions areconstrained to varying degrees by their intra- and inter-molecularassociations. For example, ligand binding sites may be covalently joinedto a single structure, noncovalently associated in a multimericstructure, embedded in a membrane or polymeric matrix, and so on andtherefore have less translational and rotational freedom than if thesame structures were present as monomers in solution.

The terms “agonism”, “partial agonism”, and “antagonism” are well knownin the art. The term “modulatory effect” refers to the ability of theligand to change the activity of an agonist or antagonist throughbinding to a ligand binding site.

The term “inert organic solvent” or “inert solvent” means a solventwhich is inert under the conditions of the reaction being described inconjunction therewith including, by way of example only, benzene,toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform,methylene chloride, diethyl ether, ethyl acetate, acetone, methylethylketone, methanol, ethanol, propanol, isopropanol, t-butanol, dioxane,pyridine, and the like. Unless specified to the contrary, the solventsused in the reactions described herein are inert solvents.

The term “treatment” refers to any treatment of a pathologic conditionin a mammal, particularly a human, and includes:

(i) preventing the pathologic condition from occurring in a subjectwhich may be predisposed to the condition but has not yet been diagnosedwith the condition and, accordingly, the treatment constitutesprophylactic treatment for the disease condition;

(ii) inhibiting the pathologic condition, i.e., arresting itsdevelopment;

(iii) relieving the pathologic condition, i.e., causing regression ofthe pathologic condition; or

(iv) relieving the conditions mediated by the pathologic condition.

The term “pathologic condition which is modulated by treatment with aligand” covers all disease states (i.e., pathologic conditions) whichare generally acknowledged in the art to be usefully treated with aligand for the β2-adrenergic receptor in general, and those diseasestates which have been found to be usefully treated by a specificmultibinding compound of our invention. Such disease states include, byway of example only, the treatment of a mammal afflicted with asthma,chronic bronchitis, chronic pulmonary obstructive disease, and the like.

The term “therapeutically effective amount” refers to that amount ofmultibinding compound which is sufficient to effect treatment, asdefined above, when administered to a mammal in need of such treatment.The therapeutically effective amount will vary depending upon thesubject and disease condition being treated, the weight and age of thesubject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art.

The term “linker”, identified where appropriate by the symbol ‘X’,refers to a group or groups that covalently attaches from 2 to 10ligands (as identified above) in a manner that provides for a compoundcapable of multivalency. Among other features, the linker is aligand-orienting entity that permits attachment of at least two copiesof a ligand (which may be the same or different) thereto. Additionally,the linker can be either a chiral or achiral molecule. In some cases,the linker maybe a covalent bond that attaches the ligands in a mannerthat provides for a compound capable of multivalency. Additionally, insome cases, the linker may itself be biologically active. The term“linker” does not, however, extend to cover solid inert supports such asbeads, glass particles, fibers, and the like. But it is understood thatthe multibinding compounds of this invention can be attached to a solidsupport if desired. For example, such attachment to solid supports canbe made for use in separation and purification processes and similarapplications.

The extent to which multivalent binding is realized depends upon theefficiency with which the linker or linkers that joins the ligandspresents these ligands to the array of available ligand binding sites.Beyond presenting these ligands for multivalent interactions with ligandbinding sites, the linker or linkers spatially constrains theseinteractions to occur within dimensions defined by the linker orlinkers. Thus, the structural features of the linker (valency, geometry,orientation, size, flexibility, chemical composition, etc.) are featuresof multibinding agents that play an important role in determining theiractivities.

The linkers used in this invention are selected to allow multivalentbinding of ligands to the ligand binding sites of a β2 adrenergicreceptor, whether such sites are located interiorly, both interiorly andon the periphery of the receptor structure, or at any intermediateposition thereof.

Representative Compounds of Formula (I):

I. Representative multibinding compounds of Formula (I)

-   wherein p is 2, q is 1, Ar¹ is 4-hydroxy-3-hydroxymethylphenyl, Ar²    is 1,4-phenylene, R¹ and R² are hydrogen, X, W, Q, and Ar are as    defined in Table A below are:

TABLE A

Stereo- Cpd. chem. at # *C W X -Q-Ar³ (** = stereochem) 1A (RS) —(CH₂)₂—bond —NH—CH₂—** CH(OH)phenyl ** = (S) 2A (RS) —(CH₂)₂— bond —NH—CH₂—**CH(OH)phenyl ** = (R) 3A (RS) —(CH₂)₂— bond —NH—CH₂—** CH(OH)phenyl ** =(RS) 4A (PS) —(CH₂)₂— bond —NH—CH₂—** CH(OH)-(4-hydroxy-3-hydroxy-methyl)phenyl ** = (RS) 5A (RS) —(CH₂)₆— bond —(CH₂)₃—O—(CH₂)₆—NH—CH₂—** CH(OH)-(4-hydroxy-3- hydroxyethyl)phenyl ** = (RS) 6A(RS) —CH₂— bond —NH—CH₂—** CH(OH)-(4-hydroxy-3- hydroxy-methyl)phenyl **= (RS) 7A (R) —(CH₂)₂— bond —NH—CH₂—** CH(OH)phenyl ** = (S) 8A (R)—(CH₂)₂— bond —NH—CH₂—** CH(OH)phenyl ** = (R) 9A (RS) —(CH₂)₆—O— bond—O—(CH₂)₆—O-[4-(3-hydroxypropyl)]- (CH₂)₃ phenyl 10A  (RS) —CH₂*CH(OH)—bond —O—(CH₂)—** CH(OH)—(CH₂)—NH—CH₂— CH₂O—* = (RS) **CH(OH)-(4-hydroxy-3-hydroxy- methyl)phenyl ** = (RS) 11A  (RS) —(CH₂)₂—bond —NH—CH₂—** CH(OH)—O-naphth-1-yl ** = (RS)II. Representative multibinding compounds of Formula (I) wherein p is 2,q is 1, Ar¹ is 4-hydroxy-3-hydroxymethylphenyl, Ar² is 1,4-phenylene, R¹and R² are hydrogen, X, W, Q, and Ar, are as defined Table B below are:

TABLE B

Stereo- Cpd. chem. at # *C W X Q —Ar³ 1B (RS) bond —O-(p-C₆H₄)—NH—CH₂—bond 4-hydroxy-3- ** CH(OH)—** = (RS) hydroxymethyl phenyl 2B (RS) bond—O— bond 4-aminophenyl 3B (RS) —(CH₂)₆— —O—(CH₂)₁₀—O-(p-C₆H₄)— bond4-hydroxy-3- O— (CH₂)₃—O—(CH₂)₆—NH— hydroxy- (CH₂)₃— CH₂—** CH(OH)—methylphenyl ** = (RS) stereochem. 4B (RS) —(CH₂)₆——O—(CH₂)₆—O-(p-C₆H₄)— bond 4-hydroxy-3- O— (CH₂)₃—O—(CH₂)₅—NH— hydroxy-(CH₂)₃— CH₂—** CH(OH)— methylphenyl ** = (RS) stereochem. 5B (RS)—(CH₂)₂— —O—(CH₂)₄— bond phenylIII. Representative multibinding compounds of Formula (I) wherein p is2, q is 1, Ar¹ is 4-hydroxy-3-hydroxy-methylphenyl, R¹ and R² arehydrogen, Ar¹ is (4-hydroxy-3-hydroxymethyl)phenyl, X, W, Q, and Ar areas defined in Table C below are:

TABLE C

Cpd. Stereochem. # at *C W X Ar² Q 1C (RS) bond bond trans-1,4-—NH—CH₂—** CH(OH)— cyclohexane ** = (RS) 2C (RS) —CH₂— bond 1,3-—CH₂—NH—CH₂—** cyclohexane CH(OH)— ** = (RS) 3C (RS) —(CH₂)₃— bond1,4-piperazine —(CH₂)₃—NH—CH₂—** CH(OH)— ** = (RS) 4C (RS) bond bondp-menthane —NH—CH₂—** CH(OH) ** = (RS) 5C (RS) bond bond 1,2-phenylene—CH₂—NH—CH₂—** CH(OH)— ** = (RS)IV. Representative multibinding compounds of Formula (I) wherein p is 2,q is 1, Ar¹ and Ar³ are 4-hydroxy-3-hydroxymethylphenyl, R¹ and R² arehydrogen, Q is a bond, and W, Ar², and X are as defined in Table D beloware:

TABLE D

Stereo- Cpd. chem. # at *C W Ar² X 1D (RS) bond 1,4-—(CH₂)-(p-C₆H₁₀)—NH—CH₂— cyclohexane ** CH(OH)— ** = (RS) stereochem.V. Representative multibinding compounds of Formula (I) wherein p is 2,q is 1, R¹ and R² are hydrogen, W is —(CH₂)₂—, Ar² is 1,4-phenylene,-Q-Ar³, is [2-hydroxy-2-phenyl]ethylamino, X is a bond and Ar¹ is asdefined below are as shown in Table E below:

TABLE E

Stereochem. Stereochem. Cpd. # Ar¹ at *C at **C 1E phenyl (RS) (RS) 2Ephenyl (R) (S) 3E phenyl (R) (R) 4E 4-amino-3,5-dichlorophenyl (RS) (RS)5E 4-amino-3,5-dichlorophenyl (R) (R) 6E 4-amino-3,5-dichlorophenyl (S)(S) 7E 4-amino-3,5-dichlorophenyl (R) (S) 8E 4-amino-3,5-dichlorophenyl(S) (R) 9E 3-formyl-amino-4-hydroxyphenyl (RS) (RS) 10E 3-formyl-amino-4-hydroxyphenyl (R) (R) 11E 3-formyl-amino-4-hydroxyphenyl (S) (S) 12E 3-formyl-amino-4-hydroxyphenyl (R) (S) 13E 3-formyl-amino-4-hydroxyphenyl (S) (R)VI. Miscellanous Compounds:

PREFERRED EMBODIMENTS

While the broadest definition of this invention is set forth in theSummary of the Invention, certain compounds of Formula (I) arepreferred.

(A) A Preferred Group is a Multibinding Compound of Formula (II):

where *C has RS, R, or S stereochemistry;Within this group (A) a more preferred group of compounds is thatwherein:

-   (i) Ar¹ is aryl, more preferably Ar¹ is a phenyl ring of formula    (c):

wherein:

R⁴ is hydrogen, alkyl, halo, or alkoxy, preferably hydrogen, methyl,fluoro, chloro, or methoxy;

R⁵ is hydrogen, hydroxy, halo, halo, amino, or —NHSO₂R^(a) where R^(a)is alkyl, preferably hydrogen, hydroxy, fluoro, chloro, amino, or—NHSO₂CH₃; and

R⁶ is hydrogen, halo, hydroxy, alkoxy, substituted alkyl, sulfonylamino,aminoacyl, or acylamino; preferably hydrogen, chloro, fluoro, hydroxy,methoxy, hydroxymethyl, —CH₂SO₂CH₃, —NHSO₂CH₃, —NHCHO, —CONH₂, or—NHCONH₂;

-   (ii) Ar¹ is heteroaryl, more preferably Ar¹ is    2,8-dihydroxyquinolin-5-yl or 3-bromoisoxazol-5-yl; or-   (iii) Ar¹ is heterocyclyl, more preferably Ar¹ is heterocyclyl fused    to an aryl ring, most preferably 6-fluorochroman-2-yl;

W is a bond linking the —NR²— group to Ar², alkylene, or a substitutedalkylene group wherein one or more of the carbon atoms in the alkyleneand the substituted alkylene group is optionally replaced by —O—,preferably a covalent bond, methylene, ethylene, propylene,—(CH₂)₆—O—(CH₂)₃—, —(CH₂)₆—O—, or —CH₂CH(OH)CH₂—O—; and

Ar² is phenyl wherein the W and the X groups are attached at the 1,2-,1,3-, and 1,4-positions of the phenyl ring; cyclohexyl optionallysubstituted with methyl and wherein the W and the X groups are attachedat the 1,3- and 1,4-positions of the cyclohexyl ring; or piperazinewherein the W and the X groups are attached at the 1,4-positions of thepiperazine ring, preferably 1,4-phenylene.

Within the above more preferred groups, even more preferred groups ofcompounds are wherein:

-   (a) X is —O—, —O-alkylene, —O-(arylene)-NH-(substituted alkylene)-,    —O-(alkylene)-O-(arylene)-(alkylene)-O-(alkylene)-NH-(substituted    alkylene)-, —O-(alkylene)-O-(arylene)-, or    -(alkylene)-(cycloalkylene)-NH-(substituted alkylene)-, preferably    —O—(CH₂)₄—; —CH₂-(1,4-cyclohexyl)-NH—CH₂—CH(OH)—;    —O-(1,4-phenylene)-NH—CH₂—CH(OH)—;    —O—(CH₂)₁₀—O-(1,4-phenylene)-(CH₂)₃—O—(CH₂)₆—NH—CH₂—CH(OH)—;    —O—(CH)₆—O-(1,4-phenylene)-(CH₂)₃—O—(CH₂)₅—NH—CH₂—CH(OH)—; or    —O—(CH₂)₆—O-(1,4-phenylene)-; and

Q is a covalent bond; or

-   (b) X is a bond; and

Q is a substituted alkylene group wherein one or more of the carbonatoms in said substituted alkylene group is optionally replaced by aheteroatom selected from the group consisting of —NR^(a)— (where R^(a)is hydrogen, alkyl, or acyl) and —O—, preferably —NH—CH₂—**CH(OH)—;—NH—CH₂—**CH(OH)—CH₂—; —NH—**CH(CH₂OH)—; —CH₂—NH—CH₂—**CH(OH)—;—C(CH₃)₂—NH—CH₂—**CH(OH)—; —(CH₂)₃—NH—CH₂—**CH(OH)—;—(CH₂)₃—(CH)₆—NH—CH₂—**CH(OH)—; —(CH₂)₂—NH—CH₂—**CH(OH)—;—O—(CH)—**CH(OH)—CH₂—NH—CH₂—**CH(OH)—; or —NH—CH₂—**CH(OH)—CH₂—O—; morepreferably —NH—CH₂—**CH(OH)—; —NH—**CH(CH₂OH)—;—(CH₂)₃—(CH₂)₆—NH—CH₂—**CH(OH)—; or —NH—CH₂—**CH(OH)—CH₂—O— (where ** isRS, R or S stereochemistry), most preferably —NH—**CH(CH₂OH)— where **is RS, R or S stereochemistry;

Within the above preferred, more preferred group of compounds, aparticularly preferred group of compounds is that wherein:

-   (i) Ar³ is same as Ar¹ as defined in preferred embodiments    (A)(i)–(iii) above.

Another particularly preferred group of compounds is that wherein:

-   (ii) Ar³ is a phenyl ring of formula (d):

wherein:

R⁷ is hydrogen, alkyl, alkenyl, substituted alkyl, halo, alkoxy,substituted alkoxy, hydroxy, aminoacyl, or heteroaryl, preferablyhydrogen, methyl, propen-2-yl, fluoro, chloro, methoxy, —OCH₂CO₂Me,—OCON(CH₃)₂, hydroxy, —CH₂CONH₂, —NHCOCH₃, —NHCHO, imidazol-1-yl, or1-methyl-4-trifluoromethylimidazol-2-yl; and

R⁸ is hydrogen, halo, alkoxy, substituted alkoxy, or acylamino,preferably hydrogen, fluoro, chloro, methoxy, —OCH₂CO₂Me, —OCON(CH₃)₂,—NHCHO, or —CONH₂.

-   (iii) Yet another particularly preferred group of compounds is that    wherein:

Ar³ is naphthyl, pyridyl, benzimidazol-1-yl, indolyl, 2-cyanoindolyl,carbazolyl, 4-methylindanyl, 5-(CH₃CO₂CH₂O—)-1,2,3,4-tetrahydronaphthyl,1H-2-oxoindole, 2,3,4-trihydrothianaphthalene,4-hydroxy-2-benzothiazolinone, or 4-oxo-2,3-dihydrothianapthalene.

Within the above preferred, more preferred, and particularly preferredgroups, even more particularly preferred group is that wherein:

Ar¹ is phenyl, 4-hydroxyphenyl, 3,4-dihydroxyphenyl, 3,4-dichlorophenyl,3,5-dihydroxyphenyl, 2-chloro-3,4-dihydroxyphenyl,2-fluoro-3,4-dihydroxyphenyl, 2-chloro-3,5-dihydroxyphenyl,2-fluoro-3,5-dihydroxyphenyl, 4-hydroxy-3-methoxyphenyl,4-hydroxy-3-hydroxymethylphenyl, 4-hydroxy-3-(HCONH—)phenyl,4-hydroxy-3-(NH₂CO—)phenyl, 3-chlorophenyl, 2,5-dimethoxyphenyl,4-(CH₃SO₂NH—)-phenyl, 4-hydroxy-3-(CH₃SO₂CH₂-)phenyl,4-hydroxy-3-(CH₃SO₂NH—)phenyl, 4-hydroxy-3-(NH₂CONH—)phenyl,3,5-dichloro-4-aminophenyl,

preferably 4-hydroxy-3-hydroxymethylphenyl, 4-hydroxy-3-(HCONH—)phenyl,3,5-dichloro-4-aminophenyl, or

most preferably 4-hydroxy-3-(HCONH—)phenyl or3,5-dichloro-4-aminophenyl; and

Ar³ is:

preferably, phenyl or 4-hydroxy-3-hydroxymethylphenyl, more preferablyphenyl.

GENERAL SYNTHETIC SCHEME

Compounds of this invention can be made by the methods depicted in thereaction schemes shown below.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCo., (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA),Emka-Chemie, or Sigma (St. Louis, Mo., USA) or are prepared by methodsknown to those skilled in the art following procedures set forth inreferences such as Fieser and Fieser's Reagents for Organic Synthesis,Volumes 1–15 (John Wiley and Sons, 1991); Rodd's Chemistry of CarbonCompounds, Volumes 1–5 and Supplementals (Elsevier Science Publishers,1989), Organic Reactions, Volumes 1–40 (John Wiley and Sons, 1991),March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition),and Larock's Comprehensive Organic Transformations (VCH Publishers Inc.,1989).

The starting materials and the intermediates of the reaction may beisolated and purified if desired using conventional techniques,including but not limited to filtration, distillation, crystallization,chromatography, and the like. Such materials may be characterized usingconventional means, including physical constants and spectral data.

Furthermore, it will be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, New York, 1991, andreferences cited therein.

These schemes are merely illustrative of some methods by which thecompounds of this invention can be synthesized, and variousmodifications to these schemes can be made and will be suggested to oneskilled in the art having referred to this disclosure.

Preparation of a Multibinding Compound of Formula (I)

In general, a multibinding compound of Formula (I) where p is 2 and q is1 can be prepared as illustrated and described in Schemes A–D below.

A multibinding compound of Formula (I) where p is 2 and q is 1 can beprepared by covalently attaching the ligands, L, wherein at least one ofthe ligand is selected from a compound of formula (a) as defined in theSummary of the Invention, to a linker, X, as shown in Scheme A below.

In method (a), a multibinding compound of Formula (I) where p is 2 and qis 1 is prepared in one step, by covalently attaching the ligands, L, toa linker, X, where FG¹ and FG² represent a functional group such ashalo, amino, hydroxy, thio, aldehyde, ketone, carboxy, carboxyderivatives such as acid halide, ester, amido, and the like. This methodis preferred for preparing compounds of Formula (I) where the ligandsare the same.

In method (b), the compounds of Formula (I) are prepared in a stepwisemanner by covalently attaching one equivalent of a ligand, L₁, with aligand X where where FG¹ and FG² represent a functional group as definedabove, and FG²PG is a protected functional group to give an intermediateof formula (II). Deprotection of the second functional group on theligand, followed by reaction with a ligand L₂, which may be same ordifferent than ligand L₁, then provides a compound of Formula (I). Thismethod is suitable for preparing compounds of Formula (I) where theligands are the non-identical.

The ligands are covalently attached to the linker using conventionalchemical techniques providing for covalent linkage of the ligand to thelinker. Reaction chemistries resulting in such linkages are well knownin the art and involve the use of complementary functional groups on thelinker and ligand as shown in Table I below.

TABLE I Representative Complementary Binding Chemistries First ReactiveGroup Second Reactive Group Linkage carboxyl amine amide sulfonyl halideamine sulfonamide hydroxyl alkyl/aryl halide ether hydroxyl isocyanatemethane amine epoxide β-hydroxyamine amine alkyl/aryl halide alkylamineamine isocyanate urea hydroxyl carboxyl ester amine aldehyde amine

Reaction between a carboxylic acid of either the linker or the ligandand a primary or secondary amine of the ligand or the linker in thepresence of suitable, well-known activating agents such asdicyclohexylcarbodiimide, results in formation of an amide bondcovalently linking the ligand to the linker; reaction between an aminegroup of either the linker or the ligand and a sulfonyl halide of theligand or the linker, in the presence of a base such as triethylamine,pyridine, an the like results in formation of a sulfonamide bondcovalently linking the ligand to the linker; and reaction between analcohol or phenol group of either the linker or the ligand and an alkylor aryl halide of the ligand or the linker in the presence of a basesuch as triethylamine, pyridine, and the like, results in formation ofan ether bond covalently linking the ligand to the linker.

A multibinding compound of Formula (I) where p is 2 q is 1, the secondligand Ar³ is the same as Ar¹, X is a bond, and Q is 2-hydroxyethylaminogroup, and the ligands are linked through the Ar² group can be preparedfrom an aryl glyoxal derivative of formula 1 as shown in Scheme B below.

Condensation of an acetophenone derivative of formula 1 with a diamineof formula 2 in an ethereal solution such as tetrahydrofuran provides animine of formula 3. Reduction of the imine with a suitable reducingagent such as diborane provides a compound of Formula (I). Suitablereaction solvents are tetrahydrofuran, and the like. Compound 1 whereAr¹ is phenyl is prepared by heating acetophenone in 48% hydrobromicacid in dimethylsulfoxide.

Compounds of formula 1 can be prepared by methods well known in the art.For example, α,α-dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone canbe prepared by heating 5-acetylsalicylic acid methyl ester in 48%hydrobromic acid.

Alternatively, a multibinding compound of Formula (I) where p is 2, q is1, the second ligand Ar³ is the same as Ar¹, X is a bond, and Q is2-hydroxyethylamino group, and the ligands are linked through the Argroup can be prepared from an aryl epoxide of formula 4 as shown inScheme C below.

A compound of Formula (I) can be prepared by reacting an epoxide offormula 4 with a diamine of formula 2. Epoxides 4 are eithercommercially available or they can be prepared by the methods describedin Kierstead, R. W. et. al. J. Med. Chem. 26, 1561–1569, (1983) or Hett,R. et. al. Tet. Lett. 35, 9345–9348 (1994).

Another method of preparing a multibinding compound of Formula (I) wherep is 2, q is 1, the second ligand Ar³ is the same as Ar¹, X is a bond,and Q is 2-hydroxyethylamino group, and the ligands are linked throughthe Ar² group can be prepared from an acetophenone derivative of formula5 as shown in Scheme D below.

Bromination of an acetophenone derivative of formula 5 with bromine in ahalogenated organic solvent such as chloroform provides anα-bromoacetophenone derivative of formula 6. Treatment of 6 with sodiumazide followed by reduction of the resulting azide 7 with a suitablereducing agent such as lithium aluminum hydride provides ethanolaminederivative of formula 8. Condensation of two equivalents of 8 with adialdehyde compound of formula 9 provides an imine of formula 10 whichis converted to a compound of Formula (I) as described in Scheme Aabove.

Another method of preparing a multibinding compound of Formula (I) wherep is 2, q is 1, Ar¹ and A² are different, X is a bond, and Q is2-hydroxyethylamino group, and the ligands are linked through the Ar²group can be prepared as shown in Scheme E below.

Condensation of a diamine of formula 11 (where PG₁ and PG₂ are suitableamino protecting groups which can be selectively removed) with a glyoxalof formula 2 followed by reduction of the resulting imine of formula 12with a suitable reducing agent such as diborane in a suitable reactionsolvents such as tetrahydrofuran provides a compound of formula 13.Compounds of formula 11 can be prepared by methods described in FIG. 14.

Reaction of compound 15 with an alpha bromoacetophenone compound offormula 6 followed by reduction of the keto group provides a compound offormula 16. The reaction is carried out under conditions well known inthe art.

Deprotection of the amino protecting group then provides a compound ofFormula (I). The deprotection reaction conditions depend on the natureof the protecting group. For example, if the protecting group is benzyl,it is removed under catalytic hydrogenation reaction conditions.

Any compound which is a β2 adrenergic receptor agonist can be used as aligand in this invention. Typically, a compound selected for use as aligand will have at least one functional group, such as an amino,hydroxyl, thiol or carboxyl group and the like, which allows thecompound to be readily coupled to the linker. Compounds having suchfunctionality are either known in the art or can be prepared by routinemodification of known compounds using conventional reagents andprocedures.

Linkers can be attached to different positions on the ligand molecule toachieve different orientations of the ligand domains, and therebyfacilitate multivalency. While a number of positions onβ-adrenergic-modulating ligands are synthetically practical for linking,it is preferred to preserve those ligand substructures which are mostimportant for ligand-receptor binding. At present, the aryl group andthe sidechain nitrogen are preferred points of attachment.

It will be apparent to one skilled in the art that the above chemistriesare not limited to preparing bivalent multibinding compounds of Formula(I) and can be used to prepare tri-, tetra-, etc., multibindingcompounds of Formula (I).

The linker is attached to the ligand at a position that retains liganddomain-ligand binding site interaction and specifically which permitsthe ligand domain of the ligand to orient itself to bind to the ligandbinding site. Such positions and synthetic protocols for linkage arewell known in the art. The term linker embraces everything that is notconsidered to be part of the ligand.

The relative orientation in which the ligand domains are displayedderives from the particular point or points of attachment of the ligandsto the linker, and on the framework geometry. The determination of whereacceptable substitutions can be made on a ligand is typically based onprior knowledge of structure-activity relationships (SAR) of the ligandand/or congeners and/or structural information about ligand-receptorcomplexes (e.g., X-ray crystallography, NMR, and the like). Suchpositions and the synthetic methods for covalent attachment are wellknown in the art Following attachment to the selected linker (orattachment to a significant portion of the linker, for example 2–10atoms of the linker), the univalent linker-ligand conjugate may betested for retention of activity in the relevant assay.

The linker, when covalently attached to multiple copies of the ligands,provides a biocompatible, substantially non-immunogenic multibindingcompound. The biological activity of the multibinding compound is highlysensitive to the valency, geometry, composition, size, flexibility orrigidity, etc. of the linker and, in turn, on the overall structure ofthe multibinding compound, as well as the presence or absence of anionicor cationic charge, the relative hydrophobicity/hydrophilicity of thelinker, and the like on the linker. Accordingly, the linker ispreferably chosen to maximize the biological activity of themultibinding compound. The linker may be chosen to enhance thebiological activity of the molecule. In general, the linker may bechosen from any organic molecule construct that orients two or moreligands to their ligand binding sites to permit multivalency. In thisregard, the linker can be considered as a “framework” on which theligands are arranged in order to bring about the desiredligand-orienting result, and thus produce a multibinding compound.

For example, different orientations can be achieved by including in theframework groups containing mono- or polycyclic groups, including aryland/or heteroaryl groups, or structures incorporating one or morecarbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylenegroups). Other groups can also include oligomers and polymers which arebranched- or straight-chain species. In preferred embodiments, rigidityis imparted by the presence of cyclic groups (e.g., aryl, heteroaryl,cycloalkyl, heterocyclic, etc.). In other preferred embodiments, thering is a six or ten member ring. In still further preferredembodiments, the ring is an aromatic ring such as, for example, phenylor naphthyl.

Different hydrophobic/hydrophilic characteristics of the linker as wellas the presence or absence of charged moieties can readily be controlledby the skilled artisan. For example, the hydrophobic nature of a linkerderived from hexamethylene diamine (H₂N(CH₂)₆NH₂) or related polyaminescan be modified to be substantially more hydrophilic by replacing thealkylene group with a poly(oxyalkylene) group such as found in thecommercially available “Jeffamines”.

Different frameworks can be designed to provide preferred orientationsof the ligands. Such frameworks may be represented by using an array ofdots (as shown below) wherein each dot may potentially be an atom, suchas C, O, N, S, P, H, F, Cl, Br, and F or the dot may alternativelyindicate the absence of an atom at that position. To facilitate theunderstanding of the framework structure, the framework is illustratedas a two dimensional array in the following diagram, although clearlythe framework is a three dimensional array in practice:

. . . . . . . . . . . . . . . . . . . . . . . . 8 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . .. 7 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . . . 6 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . . . 5 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪▪ . . . 4 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . . . 3 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . . . 2 ▪ ▪ ▪ ▪ ▪▪ ▪ ▪ ▪ . . . 1 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . . . 0 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ . . . 0 1 23 4 5 6 7 8

Each dot is either an atom, chosen from carbon, hydrogen, oxygen,nitrogen, sulfur, phosphorus, or halogen, or the dot represents a pointin space (i.e., an absence of an atom). As is apparent to the skilledartisan, only certain atoms on the grid have the ability to act as anattachment point for the ligands, namely, C, O. N, S and P.

Atoms can be connected to each other via bonds (single, double or triplebonds with acceptable resonance and tautomeric forms), with regard tothe usual constraints of chemical bonding. Ligands may be attached tothe framework via single, double or triple bonds (with chemicallyacceptable tautomeric and resonance forms). Multiple ligand groups (2 to10) can be attached to the framework such that the minimal, shortestpath distance between adjacent ligand groups does not exceed 100 atoms.Preferably, the linker connections to the ligand is selected such thatthe maximum spatial distance between two adjacent ligands is no morethan 100 Å.

An example of a linker as presented by the grid is shown below for abiphenyl construct.

Nodes (1,2), (2,0), (4,4), (5,2), (4,0), (6,2), (7,4), (9,4), (10,2),(9,0), (7,0) all represent carbon atoms. Node (10,0) represents achlorine atom. All other nodes (or dots) are points in space (i.e.,represent an absence of atoms).

Nodes (1,2) and (9,4) are attachment points. Hydrogen atoms are affixedto nodes (2,4), (4,4), (4,0), (2,0), (7,4), (10,2) and (7,0). Nodes(5,2) and (6,2) are connected by a single bond.

The carbon atoms present are connected by either a single or doublebonds, taking into consideration the principle of resonance and/ortautomerism.

The intersection of the framework (linker) and the ligand group, andindeed, the framework (linker) itself can have many different bondingpatterns. Examples of acceptable patterns of three contiguous atomarrangements are shown in the following diagram:

CCC NCC OCC SCC PCC CCN NCN OCN SCN PCN CCO NCO OCO SCO PCO CCS NCS OCSSCS PCS CCP NCP OCP SCP PCP CNC NNC ONC SNC PNC CNN NNN ONN SNN PNN CNONNO ONO SNO PNO CNS NNS ONS SNS PNS CNP NNP ONP SNP PNP COC NOC OOC SOCPOC COO NON OON SON PON COC NOO OOO SOO POO COP NOP OOS SOS POS CSC NSCOOP SOP POP CSN NSN OSC SSC PSC CSO NSO OSN SSN PSN CSS NSS OSO SSO PSOCSP NSP OSS SSS PSS CPC NPC OSP SSP PSP CPN NPN OPC SPC PPC CPO NPO OPNSPN PPN CPS NPS OPO SPO PPO CPP NPP OPS SPS PPS OPP SPP PPP

One skilled in the art would be able to identify bonding patterns thatwould produce multivalent compounds. Methods for producing these bondingarrangements are described in March, “Advanced Organic Chemistry”, 4thEdition, Wiley-Interscience, New York, N.Y. (1992). These arrangementsare described in the grid of dots shown in the scheme above. All of thepossible arrangements for the five most preferred atoms are shown. Eachatom has a variety of acceptable oxidation states. The bondingarrangements underlined are less acceptable and are not preferred.

Examples of molecular structures in which the above bonding patternscould be employed as components of the linker are shown below.

identification of an appropriate framework geometry and size for liganddomain presentation are important steps in the construction of amultibinding compound with enhanced activity. Systematic spatialsearching strategies can be used to aid in the identification ofpreferred frameworks through an iterative process. FIG. 3 illustrates auseful strategy for determining an optimal framework display orientationfor ligand domains. Various other strategies are known to those skilledin the art of molecular design and can be used for preparing compoundsof this invention.

As shown in FIG. 1, display vectors around similar central corestructures such as a phenyl structure (Panel A) and a cyclohexanestructure (Panel B) can be varied, as can the spacing of the liganddomain from the core structure (i.e., the length of the attachingmoiety). It is to be noted that core structures other than those shownhere can be used for determining the optimal framework displayorientation of the ligands. The process may require the use of multiplecopies of the same central core structure or combinations of differenttypes of display cores.

The above-described process can be extended to trimers (FIG. 2) andcompound of higher valency (FIGS. 3 and 4).

Assays of each of the individual compounds of a collection generated asdescribed above will lead to a subset of compounds with the desiredenhanced activities (e.g., potency, selectivity, etc.). The analysis ofthis subset using a technique such as Ensemble Molecular Dynamics willprovide a framework orientation that favors the properties desired. Awide diversity of linkers is commercially available (see, e.g.,Available Chemical Directory (ACD)). Many of the linkers that aresuitable for use in this invention fall into this category. Other can bereadily synthesized by methods well known in the art and/or aredescribed below.

Having selected a preferred framework geometry, the physical propertiesof the linker can be optimized by varying the chemical compositionthereof. The composition of the linker can be varied in numerous ways toachieve the desired physical properties for the multibinding compound.

It can therefore be seen that there is a plethora of possibilities forthe composition of a linker. Examples of linkers include aliphaticmoieties, aromatic moieties, steroidal moieties, peptides, and the like.Specific examples are peptides or polyamides, hydrocarbons, aromaticgroups, ethers, lipids, cationic or anionic groups, or a combinationthereof.

Examples are given below, but it should be understood that variouschanges may be made and equivalents may be substituted without departingfrom the true spirit and scope of the invention. For example, propertiesof the linker can be modified by the addition or insertion of ancillarygroups into or onto the linker, for example, to change the solubility ofthe multibinding compound (in water, fats, lipids, biological fluids,etc.), hydrophobicity, hydrophilicity, linker flexibility, antigenicity,stability, and the like. For example, the introduction of one or morepoly(ethylene glycol) (PEG) groups onto or into the linker enhances thehydrophilicity and water solubility of the multibinding compound,increases both molecular weight and molecular size and, depending on thenature of the unPEGylated linker, may increase the in vivo retentiontime. Further PEG may decrease antigenicity and potentially enhances theoverall rigidity of the linker.

Ancillary groups which enhance the water solubility/hydrophilicity ofthe linker and, accordingly, the resulting multibinding compounds areuseful in practicing this invention. Thus, it is within the scope of thepresent invention to use ancillary groups such as, for example, smallrepeating units of ethylene glycols, alcohols, polyols (e.g., glycerin,glycerol propoxylate, saccharides, including mono-, oligosaccharides,etc.), carboxylates (e.g., small repeating units of glutamic acid,acrylic acid, etc.), amines (e.g., tetraethylenepentamine), and thelike) to enhance the water solubility and/or hydrophilicity of themultibinding compounds of this invention. In preferred embodiments, theancillary group used to improve water solubility/hydrophilicity will bea polyether.

The incorporation of lipophilic ancillary groups within the structure ofthe linker to enhance the lipophilicity and/or hydrophobicity of themultibinding compounds described herein is also within the scope of thisinvention. Lipophilic groups useful with the linkers of this inventioninclude, by way of example only, aryl and heteroaryl groups which, asabove, may be either unsubstituted or substituted with other groups, butare at least substituted with a group which allows their covalentattachment to the linker. Other lipophilic groups useful with thelinkers of this invention include fatty acid derivatives which do notform bilayers in aqueous medium until higher concentrations are reached.

Also within the scope of this invention is the use of ancillary groupswhich result in the multibinding compound being incorporated or anchoredinto a vesicle or other membranous structure such as a liposome or amicelle. The term “lipid” refers to any fatty acid derivative that iscapable of forming a bilayer or a micelle such that a hydrophobicportion of the lipid material orients toward the bilayer while ahydrophilic portion orients toward the aqueous phase. Hydrophiliccharacteristics derive from the presence of phosphato, carboxylic,sulfato, amino, sulfhydryl, nitro and other like groups well known inthe art. Hydrophobicity could be conferred by the inclusion of groupsthat include, but are not limited to, long chain saturated andunsaturated aliphatic hydrocarbon groups of up to 20 carbon atoms andsuch groups substituted by one or more aryl, heteroaryl, cycloalkyl,and/or heterocyclic group(s). Preferred lipids are phosphglycerides andsphingolipids, representative examples of which includephosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoyl-phosphatidylcholine, distearoyl-phosphatidylcholine ordilinoleoylphosphatidyl-choline could be used. Other compounds lackingphosphorus, such as sphingolipid and glycosphingolipid families are alsowithin the group designated as lipid. Additionally, the amphipathiclipids described above may be mixed with other lipids includingtriglycerides and sterols.

The flexibility of the linker can be manipulated by the inclusion ofancillary groups which are bulky and/or rigid. The presence of bulky orrigid groups can hinder free rotation about bonds in the linker or bondsbetween the linker and the ancillary group(s) or bonds between thelinker and the functional groups. Rigid groups can include, for example,those groups whose conformational lability is restrained by the presenceof rings and/or multiple bonds within the group, for example, aryl,heteroaryl, cycloalkyl, cycloalkenyl, and heterocyclic groups. Othergroups which can impart rigidity include polypeptide groups such asoligo- or polyproline chains.

Rigidity can also be imparted electrostatically. Thus, if the ancillarygroups are either positively or negatively charged, the similarlycharged ancillary groups will force the presenter linker into aconfiguration affording the maximum distance between each of the likecharges. The energetic cost of bringing the like-charged groups closerto each other will tend to hold the linker in a configuration thatmaintains the separation between the like-charged ancillary groups.Further ancillary groups bearing opposite charges will tend to beattracted to their oppositely charged counterparts and potentially mayenter into both inter- and intramolecular ionic bonds. This non-covalentmechanism will tend to hold the linker into a conformation which allowsbonding between the oppositely charged groups. The addition of ancillarygroups which are charged, or alternatively, bear a latent charge whendeprotected, following addition to the linker, include deprotonation ofa carboxyl, hydroxyl, thiol or amino group by a change in pH, oxidation,reduction or other mechanisms known to those skilled in the art whichresult in removal of the protecting group, is within the scope of thisinvention.

Rigidity may also be imparted by internal hydrogen bonding or byhydrophobic collapse.

Bulky groups can include, for example, large atoms, ions (e.g., iodine,sulfur, metal ions, etc.) or groups containing large atoms, polycyclicgroups, including aromatic groups, non-aromatic groups and structuresincorporating one or more carbon-carbon multiple bonds (i.e., alkenesand alkynes). Bulky groups can also include oligomers and polymers whichare branched- or straight-chain species. Species that are branched areexpected to increase the rigidity of the structure more per unitmolecular weight gain than are straight-chain species.

In preferred embodiments, rigidity is imparted by the presence of cyclicgroups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). Inother preferred embodiments, the linker comprises one or moresix-membered rings. In still further preferred embodiments, the ring isan aryl group such as, for example, phenyl or naphthyl.

In view of the above, it is apparent that the appropriate selection of alinker group providing suitable orientation, restricted/unrestrictedrotation, the desired degree of hydrophobicity/hydrophilicity, etc. iswell within the skill of the art. Eliminating or reducing antigenicityof the multibinding compounds described herein is also within the scopeof this invention. In certain cases, the antigenicity of a multibindingcompound may be eliminated or reduced by use of groups such as, forexample, poly(ethylene glycol).

As explained above, the multibinding compounds described herein comprise2–10 ligands attached to a linker that attaches the ligands in such amanner that they are presented to the enzyme for multivalentinteractions with ligand binding sites thereon/therein. The linkerspatially constrains these interactions to occur within dimensionsdefined by the linker. This and other factors increases the biologicalactivity of the multibinding compound as compared to the same number ofligands made available in monobinding form.

The compounds of this invention are preferably represented by theempirical Formula (L)_(p)(X)_(q) where L, X, p and q are as definedabove. This is intended to include the several ways in which the ligandscan be linked together in order to achieve the objective ofmultivalency, and a more detailed explanation is described below.

As noted previously, the linker may be considered as a framework towhich ligands are attached. Thus, it should be recognized that theligands can be attached at any suitable position on this framework, forexample, at the termini of a linear chain or at any intermediateposition.

The simplest and most preferred multibinding compound is a bivalentcompound which can be represented as L-X-L, where each L isindependently a ligand which may be the same or different and each X isindependently the linker. Examples of such bivalent compounds areprovided in FIG. 1 where each shaded circle represents a ligand. Atrivalent compound could also be represented in a linear fashion, i.e.,as a sequence of repeated units L-X-L-X-L, in which L is a ligand and isthe same or different at each occurrence, as can X. However, a trimercan also be a radial multibinding compound comprising three ligandsattached to a central core, and thus represented as (L)₃X, where thelinker X could include, for example, an aryl or cycloalkyl group.Illustrations of trivalent and tetravalent compounds of this inventionare found in FIGS. 2 and 3 respectively where, again, the shaded circlesrepresent ligands. Tetravalent compounds can be represented in a lineararray, e.g.,L-X-L-X-L-X-Lin a branched array, e.g.,

(a branched construct analogous to the isomers of butane—n-butyl,iso-butyl, sec-butyl, and t-butyl) or in a tetrahedral array, e.g.,

where X and L are as defined herein. Alternatively, it could berepresented as an alkyl, aryl or cycloalkyl derivative as above withfour (4) ligands attached to the core linker.

The same considerations apply to higher multibinding compounds of thisinvention containing 5–10 ligands as illustrated in FIG. 4 where, asbefore, the shaded circles represent ligands. However, for multibindingagents attached to a central linker such as aryl or cycloalkyl, there isa self-evident constraint that there must be sufficient attachment siteson the linker to accommodate the number of ligands present; for example,a benzene ring could not directly accommodate more than 6 ligands,whereas a multi-ring linker (e.g., biphenyl) could accommodate a largernumber of ligands.

The above described compounds may alternatively be represented as cyclicchains of the form:

and variants thereof.

All of the above variations are intended to be within the scope of theinvention defined by the Formula (L)_(p)(X)_(q).

With the foregoing in mind, a preferred linker may be represented by thefollowing formula:—X^(a)-Z-(Y^(a)-Z)_(m)-X^(a)—wherein

m is an integer of from 0 to 20;

X^(a) at each separate occurrence is selected from the group consistingof —O—, —S—, —NR—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NR—, —NRC(O)—, C(S),—C(S)O—, —C(S)NR—, —NRC(S)—, or a covalent bond where R is as definedbelow;

Z at each separate occurrence is selected from the group consisting ofalkylene, substituted alkylene, cycloalkylene, substitutedcylcoalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, cycloalkenylene, substituted cycloalkenylene,arylene, heteroarylene, heterocyclene, or a covalent bond;

each Y^(a) at each separate occurrence is selected from the groupconsisting of —O—, —C(O)—, —OC(O)—, —C(O)O—, —NR—, —S(O)_(n)—,—C(O)NR′—, —NR′C(O)—, —NR′C(O)NR′—, —NR′C(S)NR′—, —C(═NR′)—NR′—,—NR′—C(═NR′)—, —OC(O)—NR′—, —NR′—C(O)—O—, —N═C(X)—NR′—,—NR′—C(X^(a))═N—, —P(O)(OR′)—O—, —O—P(O)(OR′)—, —S(O)_(n)CR′R″—,—S(O)_(n)—NR′—, —NR′—S(O)_(n)—, —S—S—, and a covalent bond; where n is0, 1 or 2; and R, R′ and R″ at each separate occurrence are selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl,aryl, heteroaryl and heterocyclic.

Additionally, the linker moiety can be optionally substituted at anyatom therein by one or more alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryland heterocyclic group.

In view of the above description of the linker, it is understood thatthe term “linker” when used in combination with the term “multibindingcompound” includes both a covalently contiguous single linker (e.g.,L-X-L) and multiple covalently non-contiguous linkers (L-X-L-X-L) withinthe multibinding compound.

Combinatorial Libraries

The methods described above lend themselves to combinatorial approachesfor identifying multimeric compounds which possess multibindingproperties.

Specifically, factors such as the proper juxtaposition of the individualligands of a multibinding compound with respect to the relevant array ofbinding sites on a target or targets is important in optimizing theinteraction of the multibinding compound with its target(s) and tomaximize the biological advantage through multivalency. One approach isto identify a library of candidate multibinding compounds withproperties spanning the multibinding parameters that are relevant for aparticular target. These parameters include: (1) the identity ofligand(s), (2) the orientation of ligands, (3) the valency of theconstruct, (4) linker length, (5) linker geometry, (6) linker physicalproperties, and (7) linker chemical functional groups.

Libraries of multimeric compounds potentially possessing multibindingproperties (i.e., candidate multibinding compounds) and comprising amultiplicity of such variables are prepared and these libraries are thenevaluated via conventional assays corresponding to the ligand selectedand the multibinding parameters desired. Considerations relevant to eachof these variables are set forth below:

Selection of Ligand(s):

A single ligand or set of ligands is (are) selected for incorporationinto the libraries of candidate multibinding compounds which library isdirected against a particular biological target or targets e.g., β2adrenergic receptor. The only requirement for the ligands chosen is thatthey are capable of interacting with the selected target(s). Thus,ligands may be known drugs, modified forms of known drugs, substructuresof known drugs or substrates of modified forms of known drugs (which arecompetent to interact with the target), or other compounds. Ligands arepreferably chosen based on known favorable properties that may beprojected to be carried over to or amplified in multibinding forms.Favorable properties include demonstrated safety and efficacy in humanpatients, appropriate PK/ADME profiles, synthetic accessibility, anddesirable physical properties such as solubility, log P, etc. However,it is crucial to note that ligands which display an unfavorable propertyfrom among the previous list may obtain a more favorable propertythrough the process of multibinding compound formation; i.e., ligandsshould not necessarily be excluded on such a basis. For example, aligand that is not sufficiently potent at a particular target so as tobe efficacious in a human patient may become highly potent andefficacious when presented in multibinding form. A ligand that is potentand efficacious but not of utility because of a non-mechanism-relatedtoxic side effect may have increased therapeutic index (increasedpotency relative to toxicity) as a multibinding compound. Compounds thatexhibit short in vivo half-lives may have extended half-lives asmultibinding compounds. Physical properties of ligands that limit theirusefulness (e.g. poor bioavailability due to low solubility,hydrophobicity, hydrophilicity) may be rationally modulated inmultibinding forms, providing compounds with physical propertiesconsistent with the desired utility.

Orientation: Selection of Ligand Attachment Points and LinkingChemistry:

Several points are chosen on each ligand at which to attach the ligandto the linker. The selected points on the ligand/linker for attachmentare functionalized to contain complementary reactive functional groups.This permits probing the effects of presenting the ligands to theirreceptor(s) in multiple relative orientations, an important multibindingdesign parameter. The only requirement for choosing attachment points isthat attaching to at least one of these points does not abrogateactivity of the ligand. Such points for attachment can be identified bystructural information when available. For example, inspection of aco-crystal structure of a protease inhibitor bound to its target allowsone to identify one or more sites where linker attachment will notpreclude the enzyme:inhibitor interaction. Alternatively, evaluation ofligand/target binding by nuclear magnetic resonance will permit theidentification of sites non-essential for ligand/target binding. See,for example, Fesik, et al., U.S. Pat. No. 5,891,643. When suchstructural information is not available, utilization ofstructure-activity relationships (SAR) for ligands will suggestpositions where substantial structural variations are and are notallowed. In the absence of both structural and SAR information, alibrary is merely selected with multiple points of attachment to allowpresentation of the ligand in multiple distinct orientations. Subsequentevaluation of this library will indicate what positions are suitable forattachment.

It is important to emphasize that positions of attachment that doabrogate the activity of the monomeric ligand may also be advantageouslyincluded in candidate multibinding compounds in the library providedthat such compounds bear at least one ligand attached in a manner whichdoes not abrogate intrinsic activity. This selection derives from, forexample, heterobivalent interactions within the context of a singletarget molecule. For example, consider a receptor antagonist ligandbound to its target receptor, and then consider modifying this ligand byattaching to it a second copy of the same ligand with a linker whichallows the second ligand to interact with the same receptor molecule atsites proximal to the antagonist binding site, which include elements ofthe receptor that are not part of the formal antagonist binding siteand/or elements of the matrix surrounding the receptor such as themembrane. Here, the most favorable orientation for interaction of thesecond ligand molecule with the receptor/matrix may be achieved byattaching it to the linker at a position which abrogates activity of theligand at the formal antagonist binding site. Another way to considerthis is that the SAR of individual ligands within the context of amultibinding structure is often different from the SAR of those sameligands in momomeric form.

The foregoing discussion focused on bivalent interactions of dimericcompounds bearing two copies of the same ligand joined to a singlelinker through different attachment points, one of which may abrogatethe binding/activity of the monomeric ligand. It should also beunderstood that bivalent advantage may also be attained withheterodimeric constructs bearing two different ligands that bind tocommon or different targets. For example, a 5HT₄ receptor antagonist anda bladder-selective muscarinic M₃ antagonist may be joined to a linkerthrough attachment points which do not abrogate the binding affinity ofthe monomeric ligands for their respective receptor sites. The dimericcompound may achieve enhanced affinity for both receptors due tofavorable interactions between the 5HT₄ ligand and elements of the M₃receptor proximal to the formal M₃ antagonist binding site and betweenthe M₃ ligand and elements of the 5HT₄ receptor proximal to the formal5HT₄ antagonist binding site. Thus, the dimeric compound may be morepotent and selective antagonist of overactive bladder and a superiortherapy for urinary urge incontinence.

Once the ligand attachment points have been chosen, one identifies thetypes of chemical linkages that are possible at those points. The mostpreferred types of chemical linkages are those that are compatible withthe overall structure of the ligand (or protected forms of the ligand)readily and generally formed, stable and intrinsically inocuous undertypical chemical and physiological conditions, and compatible with alarge number of available linkers. Amide bonds, ethers, amines,carbamates, ureas, and sulfonamides are but a few examples of preferredlinkages.

Linkers: Spanning Relevant Multibinding Parameters Through Selection ofValency, Linker Length, Linker Geometry Rigidity, Physical Properties,and Chemical Functional Groups

In the library of linkers employed to generate the library of candidatemultibinding compounds, the selection of linkers employed in thislibrary of linkers takes into consideration the following factors:

Valency:

In most instances the library of linkers is initiated with divalentlinkers. The choice of ligands and proper juxtaposition of two ligandsrelative to their binding sites permits such molecules to exhibit targetbinding affinities and specificities more than sufficient to conferbiological advantage. Furthermore, divalent linkers or constructs arealso typically of modest size such that they retain the desirablebiodistribution properties of small molecules.

Linker Length;

Linkers are chosen in a range of lengths to allow the spanning of arange of inter-ligand distances that encompass the distance preferablefor a given divalent interaction. In some instances the preferreddistance can be estimated rather precisely from high-resolutionstructural information of targets, typically enzymes and solublereceptor targets. In other instances where high-resolution structuralinformation is not available (such as 7TM G-protein coupled receptors),one can make use of simple models to estimate the maximum distancebetween binding sites either on adjacent receptors or at differentlocations on the same receptor. In situations where two binding sitesare present on the same target (or target subunit for multisubunittargets), preferred linker distances are 2–20 Å, with more preferredlinker distances of 3–12 Å. In situations where two binding sites resideon separate (e.g., protein) target sites, preferred linker distances are20–100 Å, with more preferred distances of 30–70 Å.

Linker Geometry and Rigidity:

The combination of ligand attachment site, linker length, linkergeometry, and linker rigidity determine the possible ways in which theligands of candidate multibinding compounds may be displayed in threedimensions and thereby presented to their binding sites. Linker geometryand rigidity are nominally determined by chemical composition andbonding pattern, which may be controlled and are systematically variedas another spanning function in a multibinding array. For example,linker geometry is varied by attaching two ligands to the ortho, meta,and para positions of a benzene ring, or in cis- or trans-arrangementsat the 1,1- vs. 1,2- vs. 1,3- vs. 1,4-positions around a cyclohexanecore or in cis- or trans-arrangements at a point of ethyleneunsaturation. Linker rigidity is varied by controlling the number andrelative energies of different conformational states possible for thelinker. For example, a divalent compound bearing two ligands joined by1,8-octyl linker has many more degrees of freedom, and is therefore lessrigid than a compound in which the two ligands are attached to the 4,4′positions of a biphenyl linker.

Linker Physical Properties:

The physical properties of linkers are nominally determined by thechemical constitution and bonding patterns of the linker, and linkerphysical properties impact the overall physical properties of thecandidate multibinding compounds in which they are included. A range oflinker compositions is typically selected to provide a range of physicalproperties (hydrophobicity, hydrophilicity, amphiphilicity,polarization, acidity, and basicity) in the candidate multibindingcompounds. The particular choice of linker physical properties is madewithin the context of the physical properties of the ligands they joinand preferably the goal is to generate molecules with favorable PK/ADMEproperties. For example, linkers can be selected to avoid those that aretoo hydrophilic or too hydrophobic to be readily absorbed and/ordistributed in vivo.

Linker Chemical Functional Groups:

Linker chemical functional groups are selected to be compatible with thechemistry chosen to connect linkers to the ligands and to impart therange of physical properties sufficient to span initial examination ofthis parameter.

Combinatorial Synthesis

Having chosen a set of n ligands (n being determined by the sum of thenumber of different attachment points for each ligand chosen) and mlinkers by the process outlined above, a library of (n!)m candidatedivalent multibinding compounds is prepared which spans the relevantmultibinding design parameters for a particular target. For example, anarray generated from two ligands, one which has two attachment points(A1, A2) and one which has three attachment points (B1, B2, B3) joinedin all possible combinations provide for at least 15 possiblecombinations of multibinding compounds:

A1-A1 A1-A2 A1-B1 A1-B2 A1-B3 A2-A2 A2-B1 A2-B2 A2-B3 B1-B1 B1-B2 B1-B3B2-B2 B2-B3 B3-B3

When each of these combinations is joined by 10 different linkers, alibrary of 150 candidate multibinding compounds results.

Given the combinatorial nature of the library, common chemistries arepreferably used to join the reactive functionalies on the ligands withcomplementary reactive functionalities on the linkers. The librarytherefore lends itself to efficient parallel synthetic methods. Thecombinatorial library can employ solid phase chemistries well known inthe art wherein the ligand and/or linker is attached to a solid support.Alternatively and preferably, the combinatorial libary is prepared inthe solution phase. After synthesis, candidate multibinding compoundsare optionally purified before assaying for activity by, for example,chromatographic-methods (e.g., HPLC).

Analysis of Array by Biochemical, Analytical, Pharmacological, andComputational Methods:

Various methods are used to characterize the properties and activitiesof the candidate multibinding compounds in the library to determinewhich compounds possess multibinding properties. Physical constants suchas solubility under various solvent conditions and logD/clogD values canbe determined. A combination of NMR spectroscopy and computationalmethods is used to determine low-energy conformations of the candidatemultibinding compounds in fluid media. The ability of the members of thelibrary to bind to the desired target and other targets is determined byvarious standard methods, which include radioligand displacement assaysfor receptor and ion channel targets, and kinetic inhibition analysisfor many enzyme targets. In vitro efficacy, such as for receptoragonists and antagonists, ion channel blockers, and antimicrobialactivity, can also be determined. Pharmacological data, including oralabsorption, everted gut penetration, other pharmacokinetic parametersand efficacy data can be determined in appropriate models. In this way,key structure-activity relationships are obtained for multibindingdesign parameters which are then used to direct future work.

The members of the library which exhibit multibinding properties, asdefined herein, can be readily determined by conventional methods. Firstthose members which exhibit multibinding properties are identified byconventional methods as described above including conventional assays(both in vitro and in vivo).

Second, ascertaining the structure of those compounds which exhibitmultibinding properties can be accomplished via art recognizedprocedures. For example, each member of the library can be encrypted ortagged with appropriate information allowing determination of thestructure of relevant members at a later time. See, for example, Dower,et al., International Patent Application Publication No. WO 93/06121;Brenner, et al., Proc. Natl. Acad. Sci., USA, 89:5181 (1992); Gallop, etal., U.S. Pat. No. 5,846,839; each of which are incorporated herein byreference in its entirety. Alternatively, the structure of relevantmultivalent compounds can also be determined from soluble and untaggedlibaries of candidate multivalent compounds by methods known in the artsuch as those described by Hindsgaul, et al., Canadian PatentApplication No. 2,240,325 which was published on Jul. 11, 1998. Suchmethods couple frontal affinity chromatography with mass spectroscopy todetermine both the structure and relative binding affinities ofcandidate multibinding compounds to receptors.

The process set forth above for dimeric candidate multibinding compoundscan, of course, be extended to trimeric candidate compounds and higheranalogs thereof.

Follow-Up Synthesis and Analysis of Additional Array(s):

Based on the information obtained through analysis of the initiallibrary, an optional component of the process is to ascertain one ormore promising multibinding “lead” compounds as defined by particularrelative ligand orientations, linker lengths, linker geometries, etc.Additional libraries can then be generated around these leads to providefor further information regarding structure to activity relationships.These arrays typically bear more focused variations in linker structurein an effort to further optimize target affinity and/or activity at thetarget (antagonism, partial agonism, etc.), and/or alter physicalproperties. By iterative redesign/analysis using the novel principles ofmultibinding design along with classical medicinal chemistry,biochemistry, and pharmacology approaches, one is able to prepare andidentify optimal multibinding compounds that exhibit biologicaladvantage towards their targets and as therapeutic agents.

To further elaborate upon this procedure, suitable divalent linkersinclude, by way of example only, those derived from dicarboxylic acids,disulfonylhalides, dialdehydes, diketones, dihalides, diisocyanates,diamines, diols, mixtures of carboxylic acids, sulfonyihalides,aldehydes, ketones, halides, isocyanates, amines and diols. In eachcase, the carboxylic acid, sulfonylhalide, aldehyde, ketone, halide,isocyanate, amine and diol functional group is reacted with acomplementary functionality on the ligand to form a covalent linkage.Such complementary functionality is well known in the art as illustratedin the following table:

Complementary Binding Chemistries

First Reactive Group Second Reactive Group Linkage hydroxyl isocyanateurethane amine epoxide β-hydroxyamine hydroxyamine sulfonyl halidesulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide etheraldehyde amine/NaCNBH₃ amine ketone amine/NaCNBH₃ amine amine isocyanateurea

The following table illustrates, by way of examples, starting materials(identified as X-1 through X-418) that can be used to prepare linkersincorporated in the multibinding compounds of this invention utilizingthe chemistry described above. For example, 1,10-decanedicarboxylicacid, X1, can be reacted with 2 equivalents of a ligand carrying anamino group in the presence of a coupling reagent such as DCC to providea bivalent multibinding compound of formula (I) wherein the ligands arelinked via 1,10-decanediamido linking group.

Representative ligands for use in this invention include, by way ofexample, L-1 and L-2 as identified above wherein L-1 is selected from acompound of formula (a) and L-2 is selected from a compound of formula(b).

Combinations of ligands (L) and linkers (X) per this invention include,by way example only, homo- and hetero-dimers wherein a first ligand isselected from L-1 and the second ligand and linker is selected from thefollowing:

L-2/X-1- L-2/X-2- L-2/X-3- L-2/X-4- L-2/X-5- L-2/X-6- L-2/X-7- L-2/X-8-L-2/X-9- L-2/X-10- L-2/X-11- L-2/X-12- L-2/X-13- L-2/X-14- L-2/X-15-L-2/X-16- L-2/X-17- L-2/X-18- L-2/X-19- L-2/X-20- L-2/X-21- L-2/X-22-L-2/X-23- L-2/X-24- L-2/X-25- L-2/X-26- L-2/X-27- L-2/X-28- L-2/X-29-L-2/X-30- L-2/X-31- L-2/X-32- L-2/X-33- L-2/X-34- L-2/X-35- L-2/X-36-L-2/X-37- L-2/X-38- L-2/X-39- L-2/X-40- L-2/X-41- L-2/X-42- L-2/X-43-L-2/X-44- L-2/X-45- L-2/X-46- L-2/X-47- L-2/X-48- L-2/X-49- L-2/X-50-L-2/X-51- L-2/X-52- L-2/X-53- L-2/X-54- L-2/X-55- L-2/X-56- L-2/X-57-L-2/X-58- L-2/X-59- L-2/X-60- L-2/X-61- L-2/X-62- L-2/X-63- L-2/X-64-L-2/X-65- L-2/X-66- L-2/X-67- L-2/X-68- L-2/X-69- L-2/X-70- L-2/X-71-L-2/X-72- L-2/X-73- L-2/X-74- L-2/X-75- L-2/X-76- L-2/X-77- L-2/X-78-L-2/X-79- L-2/X-80- L-2/X-81- L-2/X-82- L-2/X-83- L-2/X-84- L-2/X-85-L-2/X-86- L-2/X-87- L-2/X-88- L-2/X-89- L-2/X-90- L-2/X-91- L-2/X-92-L-2/X-93- L-2/X-94- L-2/X-95- L-2/X-96- L-2/X-97- L-2/X-98- L-2/X-99-L-2/X-100- L-2/X-101- L-2/X-102- L-2/X-103- L-2/X-104- L-2/X-105-L-2/X-106- L-2/X-107- L-2/X-108- L-2/X-109- L-2/X-110- L-2/X-111-L-2/X-112- L-2/X-113- L-2/X-114- L-2/X-115- L-2/X-116- L-2/X-117-L-2/X-118- L-2/X-119- L-2/X-120- L-2/X-121- L-2/X-122- L-2/X-123-L-2/X-124- L-2/X-125- L-2/X-126- L-2/X-127- L-2/X-128- L-2/X-129-L-2/X-130- L-2/X-131- L-2/X-132- L-2/X-133- L-2/X-134- L-2/X-135-L-2/X-136- L-2/X-137- L-2/X-138- L-2/X-139- L-2/X-140- L-2/X-141-L-2/X-142- L-2/X-143- L-2/X-144- L-2/X-145- L-2/X-146- L-2/X-147-L-2/X-148- L-2/X-149- L-2/X-150- L-2/X-151- L-2/X-152- L-2/X-153-L-2/X-154- L-2/X-155- L-2/X-156- L-2/X-157- L-2/X-158- L-2/X-159-L-2/X-160- L-2/X-161- L-2/X-162- L-2/X-163- L-2/X-164- L-2/X-165-L-2/X-166- L-2/X-167- L-2/X-168- L-2/X-169- L-2/X-170- L-2/X-171-L-2/X-172- L-2/X-173- L-2/X-174- L-2/X-175- L-2/X-176- L-2/X-177-L-2/X-178- L-2/X-179- L-2/X-180- L-2/X-181- L-2/X-182- L-2/X-183-L-2/X-184- L-2/X-185- L-2/X-186- L-2/X-187- L-2/X-188- L-2/X-189-L-2/X-190- L-2/X-191- L-2/X-192- L-2/X-193- L-2/X-194- L-2/X-195-L-2/X-196- L-2/X-197- L-2/X-198- L-2/X-199- L-2/X-200- L-2/X-201-L-2/X-202- L-2/X-203- L-2/X-204- L-2/X-205- L-2/X-206- L-2/X-207-L-2/X-208- L-2/X-209- L-2/X-210- L-2/X-211- L-2/X-212- L-2/X-213-L-2/X-214- L-2/X-215- L-2/X-216- L-2/X-217- L-2/X-218- L-2/X-219-L-2/X-220- L-2/X-221- L-2/X-222- L-2/X-223- L-2/X-224- L-2/X-225-L-2/X-226- L-2/X-227- L-2/X-228- L-2/X-229- L-2/X-230- L-2/X-231-L-2/X-232- L-2/X-233- L-2/X-234- L-2/X-235- L-2/X-236- L-2/X-237-L-2/X-238- L-2/X-239- L-2/X-240- L-2/X-241- L-2/X-242- L-2/X-243-L-2/X-244- L-2/X-245- L-2/X-246- L-2/X-247- L-2/X-248- L-2/X-249-L-2/X-250- L-2/X-251- L-2/X-252- L-2/X-253- L-2/X-254- L-2/X-255-L-2/X-256- L-2/X-257- L-2/X-258- L-2/X-259- L-2/X-260- L-2/X-261-L-2/X-262- L-2/X-263- L-2/X-264- L-2/X-265- L-2/X-266- L-2/X-267-L-2/X-268- L-2/X-269- L-2/X-270- L-2/X-271- L-2/X-272- L-2/X-273-L-2/X-274- L-2/X-275- L-2/X-276- L-2/X-277- L-2/X-278- L-2/X-279-L-2/X-280- L-2/X-281- L-2/X-282- L-2/X-283- L-2/X-284- L-2/X-285-L-2/X-286- L-2/X-287- L-2/X-288- L-2/X-289- L-2/X-290- L-2/X-291-L-2/X-292- L-2/X-293- L-2/X-294- L-2/X-295- L-2/X-296- L-2/X-297-L-2/X-298- L-2/X-299- L-2/X-300- L-2/X-301- L-2/X-302- L-2/X-303-L-2/X-304- L-2/X-305- L-2/X-306- L-2/X-307- L-2/X-308- L-2/X-309-L-2/X-310- L-2/X-311- L-2/X-312- L-2/X-313- L-2/X-314- L-2/X-315-L-2/X-316- L-2/X-317- L-2/X-318- L-2/X-319- L-2/X-320- L-2/X-321-L-2/X-322- L-2/X-323- L-2/X-324- L-2/X-325- L-2/X-326- L-2/X-327-L-2/X-328- L-2/X-329- L-2/X-330- L-2/X-331- L-2/X-332- L-2/X-333-L-2/X-334- L-2/X-335- L-2/X-336- L-2/X-337- L-2/X-338- L-2/X-339-L-2/X-340- L-2/X-341- L-2/X-342- L-2/X-343- L-2/X-344- L-2/X-345-L-2/X-346- L-2/X-347- L-2/X-348- L-2/X-349- L-2/X-350- L-2/X-351-L-2/X-352- L-2/X-353- L-2/X-354- L-2/X-355- L-2/X-356- L-2/X-357-L-2/X-358- L-2/X-359- L-2/X-360- L-2/X-361- L-2/X-362- L-2/X-363-L-2/X-364- L-2/X-365- L-2/X-366- L-2/X-367- L-2/X-368- L-2/X-369-L-2/X-370- L-2/X-371- L-2/X-372- L-2/X-373- L-2/X-374- L-2/X-375-L-2/X-376- L-2/X-377- L-2/X-378- L-2/X-379- L-2/X-380- L-2/X-381-L-2/X-382- L-2/X-383- L-2/X-384- L-2/X-385- L-2/X-386- L-2/X-387-L-2/X-388- L-2/X-389- L-2/X-390- L-2/X-391- L-2/X-392- L-2/X-393-L-2/X-394- L-2/X-395- L-2/X-396- L-2/X-397- L-2/X-398- L-2/X-399-L-2/X-400- L-2/X-401- L-2/X-402- L-2/X-403- L-2/X-404- L-2/X-405-L-2/X-406- L-2/X-407- L-2/X-408- L-2/X-409- L-2/X-410- L-2/X-411-L-2/X-412- L-2/X-413- L-2/X-414- L-2/X-415- L-2/X-416- L-2/X-417-L-2/X-418-and so on.

Utility, Testing, and Administration Utility

The multibinding compounds of this invention are β2 adrenergic receptoragonists or partial agonists. Accordingly, the multibinding compoundsand pharmaceutical compositions of this invention are useful in thetreatment and prevention of diseases mediated by β2 adrenergic receptorsuch as asthma, chronic obstructive pulmonary disease, bronchitis, andthe like. They are also useful in the treatment of nervous system injuryand premature labor. It is also contemplated that the compounds of thisinvention are useful for treating metabolic disorders such as obesity,diabetes, and the like.

Testing

The β2 adrenergic receptor agonistic activity of the compounds ofFormula (I) to may be demonstrated by a variety of in vitro assays knownto those of ordinary skill in the art, such as the assay described inthe biological Examples 1 and 2. It may also be assayed by the ex vivoassays described in Ball, D. I. et al., “Salmeterol a Novel, Long-actingbeta 2-Adrenergic Agonist: Characterization of Pharmacological Activityin Vitro and in Vivo” Br. J. Pharmacol., 104, 665–671 (1991); Linden, A.et al., “Salmeterol, Formoterol, and Salbutamol in the IsolatedGuinea-Pig Trachea: Differences in Maximum Relaxant Effect and Potencybut not in Functional Atagonism. Thorax, 48, 547–553, (1993); and Bials,A. T. et al., Investigations into Factors Determining the Duration ofAction of the Beta 2-Adrenoceptor Agonist, Salmeterol. Br. J.Pharmacol., 108, 505–515 (1993); or in vivo assays such as thosedescribed in Ball, D. I. et al., “Salmeterol a Novel, Long-acting beta2-Adrenergic Agonist: Characterization of Pharmacological Activity inVitro and in Vivo” Br. J. Pharmacol., 104, 665–671 (1991); Kikkawa, H.et al., “TA-2005, a Novel, Long-acting, and Selective Beta2-Adrenoceptor Agonist: Characterization of its in vivo BronchodilatingAction in Guinea Pigs and Cats in Comparison with other Beta2-Agonists”. Biol. Pharm. Bull., 17, 1047–1052, (1994); and Anderson, G.P., “Formoterol: Pharmacology, Molecular basis of Agonism and Mechanismof Long Duration of a Highly Potent and Selective Beta 2-AdrenoceptorAgonist Bronchodilator, Life Sciences, 52, 2145–2160, (1993).

Pharmaceutical Formulations

When employed as pharmaceuticals, the compounds of this invention areusually administered in the form of pharmaceutical compositions. Thesecompounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andinhalation (e.g., intranasal or oral inhalation). These compounds areeffective as injectable, inhaled and oral compositions. Suchcompositions are prepared in a manner well known in the pharmaceuticalart and comprise at least one active compound. A preferred manner foradministering compounds of this invention is inhalation. This is aneffective means for delivering a therapeutic agent directly to therespiratory tract for the treatment of diseases such as asthma and othersimilar or related respiratory tract disorders (see U.S. Pat. No.5,607,915).

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds described hereinassociated with pharmaceutically acceptable carriers. In making thecompositions of this invention, the active ingredient is usually mixedwith an excipient, diluted by an excipient or enclosed within such acarrier which can be in the form of a capsule, sachet, paper or othercontainer. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form. Theterm “unit dosage forms” refers to physically discrete units suitable asunitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical excipient. Preferably, the compound of Formula (I) aboveis employed at no more than about 20 weight percent of thepharmaceutical composition, more preferably no more than about 15 weightpercent, with the balance being pharmaceutically inert carrier(s).

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. Forexample, when the drug is administered via inhalation, each dosagecontains from about 1 μg to about 1000 μg, preferably about 2 μg toabout 500 μg, more preferably about 5 μg to about 100 μg, even morepreferably about 5 μg to about 60 μg, of the active ingredient. It, willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.Furthermore, the compound of this invention may be administeredprophylactically, for example, a pharmaceutical composition containing acompound of this invention may be administered before the bronchospasmbegins in an asthma attack, to prevent its occurrence or to reduce theextent to which it occurs.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepre-formulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid pre-formulation isthen subdivided into unit dosage forms of the type described abovecontaining the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders (see U.S. Pat. No. 5,983,956). Theliquid or solid compositions may contain suitable pharmaceuticallyacceptable excipients as described supra. Preferably the compositionsare administered by the oral or nasal respiratory route for local orsystemic effect. Compositions in preferably pharmaceutically acceptablesolvents may be nebulized by use of inert gases. Nebulized solutions maybe inhaled directly from the nebulizing device or the nebulizing devicemay be attached to a face mask tent, or intermittent positive pressurebreathing machine. Solution, suspension, or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner (see U.S. Pat. Nos. 5,919,827and 5,972,919).

Furthermore, the pharmaceutical compositions containing one or morecompound(s) of this invention can be administered in combination anyother suitable drug, for example, with a suitable steroidalanti-inflammatory drug, e.g., budesonide, flucatisone, beclamethasone,for the treatment of respiratory disorders. When the combination therapyis employed, the pharmaceutical composition containing the compound(s)of this invention and the steroidal anti-inflammatory drug may beadministered simultaneously, sequentially or separately. Each componentused in the combination therapy is employed in an amount sufficient forits intended purpose. For example, the steriodal anti-inflammatory drugsare employed in sufficient amounts to effect reduction in inflammationin vivo. The β-2 adrenergic receptor agonist/partial agonist compoundsof this invention are employed in an amount sufficient to causerelaxation of smooth muscle tissue, for example, in the bronchialsystem.

Preferably, the dose range for compounds of this invention is from about1 μg to about 1000 μg per dose, more preferably about 2 μg to about 500μg, even more preferably about 5 μg to about 100 μg, and still morepreferably about 5 μg to about 60 μg. The preferred dosage range for asteroidal anti-inflammatory drug is from about 50 to 4800 μg and morepreferably about 100 μg to about 1600 μg. Again, the particular doseused will depend on the patient (age, weight, etc.), and the severity ofthe disease (mild, moderate, severe). Lastly, a pharmaceuticalcomposition containing the two active ingredients can also be preparedfor administering the drugs simultaneously.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

In the examples below, the following abbreviations have the followingmeanings. Unless otherwise stated, all temperatures are in degreesCelsius. If an abbreviation is not defined, it has its generallyaccepted meaning.

Å=Angstroms

cm=centimeter

DCC=dicyclohexyl carbodiimide

DMF=N,N-dimethylformamide

DMSO=dimethylsulfoxide

g=gram

HPLC=high performance liquid chromatography

MEM=minimal essential medium

mg=milligram

MIC=minimum inhibitory concentration

min=minute

mL=milliliter

mm=millimeter

mmol=millimol

N=normal

THF=tetrahydrofuran

μL=microliters

μm=microns

rt=room temperature

R_(f)=retention faction

NMR=nuclear magnetic resonance

ESMS=electrospray mass spectrum

ppm=parts per million

SYNTHETIC EXAMPLES Example 1 Synthesis oftrans-1,4-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}cyclohexaneFollowing FIG. 5

Step 1

To a solution of 5-acetylsalicylic acid methyl ester 11 (5.0 g, 25.7mmole) in dimethylsulfoxide (44 mL) was added 48% hydrobromic acid. Theresulting mixture was stirred at 55° C. for 24 h, and poured into aslurry of ice-water (200 mL), precipitating a pale yellow solid. Thesolid was filtered, washed with water (200 mL), and dried to giveα,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone 12. The productwas re-suspended in ethyl ether (200 mL), filtered and dried to give(3.41 g, 59%) of pure product. R_(f)=0.8 (10% MeOH/CH₂Cl₂).

H¹-NMR (4/1 CDCl₃/CD₃OD, 299.96 MHz): δ (ppm) 8.73–8.72 (d, 1H),8.28–8.24 (dd, 1H), 7.08–7.05 (d, 1H), 5.82 (s, 1H), 4.01 (s, 3H).

Step 2

To a suspension ofα,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone 12 (0.3 g, 1.33mmole) in THF (10 mL) was added a solution oftrans-1,4-diaminocyclohexane (76 mg, 0.66 mmole) in THF (5 mL). Theresulting suspension was stirred for 3 h at ambient temperature undernitrogen atmosphere, at which formation of an imine was completed judgedby TLC analysis. After cooling of the resulting solution at ice bath, anexcess amount of 2M BH₃-Me₂S in hexane (4 mL, 8 mmole) was added to theprevious solution. The resulting mixture was slowly warmed to rt andrefluxed for 4 h under N₂ stream. After cooling the reaction mixture,MeOH (5 mL) was added to quench excess amount of 2M BH₃-Me₂S. Afterstirring for 30 min., the final solution (or cloudy solution) wasevaporated in vacuo, yielding a pale brown solid. The solid was washedwith EtOAc/hexane (1/2; 20 mL), and dried. The crude product wasdissolved in 50% MeCN/H₂O containing 0.5% TFA, and purified byprep-scale high performance liquid chromatography (HPLC) using a lineargradient (5% to 50% MeCN/H₂O over 50 min, 20 mL/min; detection at 254nM). Fractions with UV absorption were analyzed by LC-MS to isolatetrans-1,4-bis{N-[2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-hydroxyethyl]amino}cyclohexane13.

H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.35 (d, 2H), 7.18 (dd, 2H),6.80–6.78 (d, 2H), 4.88–4.86 (m, 2H), 4.65 (s, 4H), 3.15 (br s, 4H),2.89 (m, 2H), 1.68–1.55 (br m, 4H); ESMS (C₂₄H₃₄N₂O₆): calcd. 446.5,obsd. 447.5 [M+H]⁺.

Compound 14:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 4,4′-methylenebis(cyclohexylamine)gave bis{4,4′-[N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]cyclohexane}methane.

ESMS (C₃₁H₄₆N₂O₆): calcd. 542.7, obsd. 543.6 [M+H]⁺.

Compound 15:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 1,3-cyclohexanebis(methylamine) gave1,3-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl}cyclohexane.

ESMS (C₂₇H₃₈N₂O): calcd. 474.6, obsd. 475.3 [M+H]⁺.

Compound 16:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 1,8-diamino-p-menthane gave 1,8-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}-p-menthane.

ESMS (C₂₈H₄₂N₂O₆): calcd. 502.6, obsd. 503.3 [M+H]⁺.

Compound 17:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 1,4-bis(3-aminopropyl)piperazine gave1,4-bis{3-[[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]propyl}piperazine.

ESMS (C₂₈H₄₄N₄O): calcd. 532.6, obsd. 533.3 [N+H]⁺, 555.0 [M+Na]⁺.

Compound 18:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with p-xylylenediamine gave1,4-bis{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]aminomethyl}benzene.ESMS (C₂₆H₃₂N₂O₆): calcd. 468.5, obsd. 469.3 [M+H]⁺, 492.0 [M+Na]⁺.

Compound 19:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with m-xylylenediamine gave1,3-bis{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]aminomethyl}benzene.ESMS (C₂₆H₃₂N₂O₆): calcd. 468.5, obsd. 469.3 [M+H]⁺, 492.0 [M+Na]⁺.

Compound 20:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 2-aminobenzylamine gave1-{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]aminomethyl}-2-{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}benzene.ESMS (C₂₅H₃₀N₂O₆): calcd. 454.5, obsd. 455.3 [M+H]⁺.

Compound 21:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 2-(4-aminophenyl)ethylamine gave1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-2-{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]benzene.ESMS (C₂₆H₃₂N₂O₆): calcd. 468.5, obsd. 469.3 [M+H]⁺.

Compound 22:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 4,4′-oxydianiline gave 4,4′-bis(N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino}phenylether.ESMS (C₃₀H₃₂N₂O₇): calcd. 532.6, obsd. 533.3 [M+H]⁺, 556.1 [M+Na]⁺.

Compound 23:

Proceeding as described above but substitutingtrans-1,4-diamino-cyclohexane with 2-aminobenzylamine gave1-{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]aminomethyl}-4-{N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino}benzene.ESMS (C₂₅H₃₁N₂O₆): calcd. 454.5, obsd. 455.5 [M+H]⁺, 477.3 [M+Na]⁺.

Example 2 Synthesis of1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-{N-[2-phenyl-2-hydroxyethyl]amino]benzeneFollowing FIG. 6

To a suspension ofα,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone 12, prepared inExample 1, Step 1 above, (0.3 g, 1.33 mmole) in THF (10 mL) was added asolution of 2-(4-aminophenyl)ethylamine 25 (0.181 g, 1.33 mmol) in THF(5 mL). The resulting suspension was stirred for 3 h at ambienttemperature under nitrogen atmosphere, followed by additionα,α-dihydroxy-acetophenone 24 (0.2 g, 1.32 mmole). The reaction mixturewas stirred for 3 h at RT, at which formation of the imine was completedas judged by TLC analysis. The reaction mixture was cooled in an icebath and an excess amount of 2M BH₃-Me₂S in hexane (9 mL; 18 mmole) wasadded. The resulting mixture was slowly warmed to rt, and refluxed for 4h under N₂ stream. After cooling, MeOH (10 mL) was added to quenchexcess amount of BH₃-Me₂S. After stirring 30 min., at rt, the finalsolution (or cloudy suspension) was evaporated in vacuo, to give a palebrown solid. The solid was washed with EtOAc/hexane (1/2; 20 mL), anddried. The crude product was dissolved in 50% MeCN/H₂O containing 0.5%TFA, and purified by prep-scale high performance liquid chromatography(HPLC) using a linear gradient (5% to 50% MeCN/H₂O over 50 min, 20mL/min; detection at 254 nM). Fractions with UV absorption were analyzedby LC-MS to locate1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]-ethyl}-4-{N-[2-phenyl-2-hydroxyethyl]amino]benzene26. ESMS (C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.3 [M+H]⁺.

Compound 27:

Proceeding as described above, but substitutingα,α-dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone withα,α-dihydroxyacetophenone gave1-{2-[N-[2-phenyl-2-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-hydroxyethyl)amino]-benzene.ESMS (C₂₄H₂₈N₂O₈): calcd. 376.5, obsd. 377.0 [M+H]⁺.

Example 3 Synthesis of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]-amino]ethyl}-4-[N-(2-phenyl-2-hydroxyethyl)amino]benzeneFollowing FIG. 7

Step 1

To a solution of 4-(2-aminoethyl)aniline 25 (20 g, 147 mmole) inmethanol (250 mL) was added (Boc)₂O (32.4 g, 148 mmole) in methanol (50mL) at rt. After stirring for 24 h, the reaction mixture wasconcentrated to dryness to afford a pale yellow oily residue. The oilymaterial solidified slowly; thus it was dissolved in 5% MeOH/CH₂Cl₂, andsubsequently applied to flash silica column chromatography (3 to 10%MeOH/CH₂Cl₂). After purification, 4-(N-Boc-2-aminoethyl)aniline 28 wasobtained as a pale yellow solid (32.95 g, 95%): R_(f)=0.6 in 10%MeOH/CH₂Cl₂. ¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm) 6.96–6.93 (d, 2H),6.69–6.65 (d, 2H), 3.20–3.13 (q, 2H), 2.63–2.58 (t, 2H), 1.41 (s, 9H).

Step 2

4-(N-Boc-2-aminoethyl)aniline 28 (1.25 g, 5.29 mmole) was dissolved inmethanol (30 mL), followed by addition of phenyl glyoxal 24 (0.708 g,5.28 mmole). The reaction mixture was stirred for 1 h at rt, prior toaddition of NaCNBH₃ (0.665 g, 10.6 mmole). The final mixture was stirredfor 12 h at rt, concentrated, and purified by flash silica columnchromatography (2 to 5% MeOH/CH₂Cl₂) to giveN-(2-phenyl-2-hydroxyethyl)-4-(N-Boc-2-aminoethyl)aniline as a paleyellow oil (1.71 g, 91%): R_(f)=0.18 in 5% MeOH/CH₂Cl₂. ¹H-NMR (CD₃OD,299.96 MHz): δ (ppm) 7.4–7.25 (m, 5H), 7.0–6.95 (d, 2H), 6.63–6.60 (d,2H), 4.85–4.79 (dd, 1H), 3.3–3.21 (t, 2H), 3.2–3.15 (m, 2H), 2.64–2.5(t, 2H), 1.42 (s, 9H).

Step 3

A solution of N-(2-phenyl-2-hydroxyethyl)-4-(N-Boc-2-aminoethyl)aniline(1.7 g, 4.77 mmole) in methylene chloride (10 mL) was cooled in icebath, and TFA (10 mL) was slowly added under a stream of nitrogen gas.The reaction mixture was stirred for 1 h, and concentrated to yield apale yellow oil. The crude material was purified by reversed phase HPLC(10% to 40% MeCN/H₂O over 50 min; 20 mL/min) to giveN-(2-phenyl-2-hydroxyethyl)-4-(2-aminoethyl)aniline 29 as the TFA salt(1.1 g). ¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.42–7.3 (m, 5H), 7.29–7.25(d, 2H), 7.12–7.0 (d, 2H), 4.85–4.82 (m, 1H), 3.45–3.35 (m, 2H),3.18–3.1 (t, 2H), 2.98–2.94 (t, 2H); ESMS (C₁₆H₂₀N₂O₁): calcd. 256.4,obsd. 257.1 [M+H]⁺, 278.8 [M+Na)⁺, 513.4 [2M+H]⁺.

Step 4

To a solution of N-(2-phenyl-2-hydroxyethyl)-4-(2-aminoethyl)anilinetrifluoroacetate salt 29 (1.1 g, 2.3 mmole) in methanol (10 mL) wasadded 5 M NaOH solution (0.93 mL). After stirring for 10 min., thesolution was concentrated to dryness. The residue was dissolved in THF(25 mL), and α,α-dihydroxy-4-hydroxy-3-methoxy-carbonylacetophenone 12(0.514 g, 2.27 mmole) was added. The reaction mixture was stirred for 12h at rt, cooled to 0° C., and BH₃/Me₂S (1.14 mL, 10 M) was added undernitrogen atmosphere. The reaction mixture was gradually warmed to rt,stirred for 2 h at rt, and refluxed for 4 h. The reaction mixture wascooled and methanol (10 mL) was added slowly. After stirring for 30min., at rt, the reaction mixture was concentrated to afford a solidresidue, which was dissolved in MeOH (20 mL) containing 10% TFA.Evaporation of the organics yielded a pale yellow oil which was purifiedby reversed phase HPLC: 10% to 30% MeCN/H₂O over 50 min; 20 mL/min togive1-{2-[N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]-amino]ethyl}-4-[N-(2-phenyl-2-hydroxyethyl)-amino]benzene30 as the TFA salt (0.65 g). ¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm)7.42–7.3 (m, 6H), 7.28–7.24 (d, 2H), 7.18–7.14 (dd, 1H), 7.1–7.07 (d,2H), 6.80–6.77 (d, 1H), 4.86–4.82 (m, 2H), 4.65 (s, 2H), 3.44–3.34 (m,2H), 3.28–3.22 (m, 2H), 3.20–3.14 (m, 2H), 3.04–2.96 (m, 2H); ESMS(C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.1 [M+H]⁺, 404.7 [M-1H₂O]⁺, 387.1[M-2H₂O]⁺.

Example 4 Synthesis of1-{2-[N-2-4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]-aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzeneFollowing FIG. 8

Step 1

A solution of 4-(N-Boc-2-aminoethyl)aniline 28 (7.0 g, 29.6 mmole) inethanol (100 mL) and (R)-styreneoxide (3.56 g, 29.6 mmole) was refluxedfor 24 h. The organics were removed to give a pale yellow solid.N-(2-phenyl-2-(S)-hydroxyethyl)-4-(N-Boc-2-aminoethyl)aniline wasseparated by flash silica column chromatography: 1/2 EtOAc/hexane to 3/1EtOAc/hexane to 3% MeOH in 3/1 EtOAc/hexane: Rf=0.39 in 3% MeOH/CH₂Cl₂.

Step 2

A solution ofN-(2-phenyl-2-(S)-hydroxyethyl)-4-(N-Boc-2-aminoethyl)aniline (2.5 g,7.0 mmole) in CH₂Cl₂ (15 mL) was cooled in an ice bath under stream ofnitrogen and TFA (15 mL) was slowly added. The reaction mixture wasstirred for 2 h at 0° C. and then concentrated in vacuo. The crudeproduct was dissolved in 20% MeCN/H₂O and purified by preparativereversed phase HPLC (5 to 2% MeCN/H₂O over 50 min; 254 nm; 20 mL/min.),to give N-(2-phenyl-2-(S)-hydroxyethyl)-4-(2-aminoethyl)anilinetrifluoroacetate salt 31 as a colorless oil.

¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm); 7.45–7.25 (m, 9H), 4.9 (dd, 1H),3.55–3.45 (m, 2H), 3.21–3.15 (t, 2H), 3.05–2.95 (t, 2H) ESMS(C₁₆H₂₀N₂O₁): calcd. 256.4, obsd. 257.1 [M+H]⁺, 280.2 [M+Na]⁺.

Step 3

To a solution of N-(2-phenyl-2-(S)-hydroxyethyl)-4-(2-aminoethyl)anilinetrifluoroacetate 31 (0.144 g, 0.3 mmole) in methanol (10 mL) was addedaq. NaOH solution (1.0 M, 0.625 mL). The solution was concentrated todryness and the residue was dissolved in anhydrous THF (5 mL).α,α-Dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone 12 (0.067 g, 0.3mmole) was added and the reaction mixture was stirred for 12 h at rt.BH₃-Me₂S (0.2 mL, 2M) was added at 0° C. and the reaction mixture washeated at 75° C. for 6 h. After cooling the reaction mixture in icebath, MeOH (5 mL) was slowly added to it to quench the reaction, and thereaction mixture was stirred for 30 min., at rt. The organics wereremoved and the residue was dissolved in TFA/MeOH (1/9; 20 mL), andconcentrated. The crude product was dissolved in 20% MeCN/H₂O, andpurified by preparative HPLC: 5 to 20% MeCN/H₂O; 20 mL/min; 254 nm.) togive1-{2-[N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]ethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)-amino]benzene33.

¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.42–7.29 (m, 8H), 7.22–7.18 (d,2H), 7.17–7.14 (dd, 1H), 6.80–6.77 (d, 1H), 4.9–4.85 (m, 2H), 4.65 (s,2H), 3.5–3.34 (m, 2H), 3.28–3.25 (m, 2H), 3.19–3.14 (m, 2H), 3.04–2.98(m, 2H); ESMS (C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.1 [M+H]⁺, 446.1[M+Na]⁺.

Proceeding as described in Example 4 above but substituting(R)-styreneoxide with (S)-styreneoxide gave1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-[N-(2-phenyl-2-(R)-hydroxyethyl)amino]benzene34.

¹H-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.42–7.28 (m, 8H), 7.20–7.1 (m, 3H),6.80–6.77 (d, 1H), 4.9–4.85 (m, 2H), 4.65 (s, 2H), 3.45–3.34 (m, 2H),3.28–3.25 (m, 2H), 3.19–3.15 (m, 2H), 3.04–2.98 (m, 2H); ESMS(C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.1 [M+H]⁺, 446.1 [M+Na]⁺.

Example 5 Synthesis of1,6-bis{4-(N-[2-4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminohexyloxypropyl]phenoxy}hexaneFollowing FIG. 9

Step 1

A solution of 3-(4-hydroxyphenyl)-1-propanol 35 (3.3 g, 21.7 mmole) and1,6-di-iodohexane (3.5 g, 8.88 mmole) in dimethylsulfoxide (40 mL) wasdegassed and saturated with N₂ gas and potassium carbonate (4.5 g, 32.56mmole) was added. The reaction mixture was stirred at 80° C. for 18 hunder nitrogen atmosphere and then quenched with brine (150 mL). Theproduct was extracted with EtOAc (200 mL) and the organic extracts werewashed with 0.1 M NaOH and brine, and dried with MgSO₄. The organicswere removed in vacuo to give a pale brown solid. The solid was purifiedby flash silica column chromatography: 4/1 hexane/EtOAc to 5% MeOH in1/1 hexane/EtOAc to give 1,6-bis[4-(3-hydroxypropyl)phenoxy]hexane 36(R_(f)=0.17 in 1/1 hexane/EtOAc) in 65% yield (2.23 g). ¹H-NMR (CD₃OD,299.96 MHz): δ (ppm) 7.08–7.05 (d, 4H), 6.80–6.77 (d, 4H), 3.93–3.89 (t,4H), 3.56–3.52 (t, 4H), 2.64–2.56 (t, 4H), 1.81–1.69 (m, 8H), 1.44–1.21(m, 4H).

Step 2

A solution of 1,6-bis[4-(3-hydroxypropyl)phenoxy]hexane 36 (2.2 g, 5.69mmole) in DMF (10 mL) was added to a solution of DMF (40 mL) containingNaH (0.57 g; 60% dispersion in mineral oil) at 0° C. under nitrogenatmosphere and the reaction mixture was heated at 50° C. After 1 h,6-bromohexanenitrile (2.26 mL, 17 mmole) was added and the reactionmixture was heated at 80° C. for 24 h. The reaction mixture was quenchedwith brine solution (100 mL) and was extracted with EtOAc (250 mL). Theorganic phase was washed with brine, dried with MgSO₄, and evaporated invacuo, to give a pale yellow oil. Purification by flash silica columnchromatography: 4/1 to 1/1 hexane/EtOAc afforded1,6-bis[4-(5-cyanopentyloxypropyl)]phenoxy]hexane 37 product (R_(f)=0.6in 1/1 EtOAc/hexane). ¹H-NMR (CDCl₃, 299.96 MHz): δ (ppm) 7.09–7.06 (d,4H), 6.82–6.79 (d, 4H), 3.94–3.90 (t, 4H), 3.42–3.37 (m, 8H), 2.64–2.58(t, 4H), 2.40–2.32 (m, 8H), 1.90–1.26 (m, 24H).

Step 3

The 1,6-bis[4-(5-cyanopentyloxypropyl)]phenoxy]hexane 37 (0.278 g, 0.48mmole) obtained in Step 2 above was added to a mixture of conc. HCl (10mL) and AcOH (2 mL) and the reaction mixture was heated at 90° C. After15 h, the reaction mixture was diluted with brine (50 mL), extractedwith EtOAc (100 mL), and dried with MgSO₄. Evaporation of the organicphase afforded the 1,6-bis[4-(5-carboxypentyl-oxypropyl)]phenoxy]hexane38 as a pale yellow oily residue, which was used in next step withoutfurther purification. ¹H-NMR (CDCl₃, 299.96 MHz): δ (ppm) 7.09–7.07 (d,4H), 6.82–6.79 (d, 4H), 3.96–3.92 (t, 4H), 3.42–3.56 (m, 8H), 2.64–2.59(t, 4H), 2.39–2.32 (m, 4H), 1.91–1.40 (m, 24H).

Step 4

To a solution of2-hydroxy-2-(4-benzyloxy-3-hydroxymethylphenyl)-ethylamine 39 (0.263 g,0.96 mmole) in DMF (8 mL) was added1,6-bis[4-(5-carboxypentyloxypropyl)phenoxy]hexane (˜0.48 mmole),obtained in Step 3 above, HOBt (0.13 g, 0.96 mmole), DIPEA (0.21 mL,1.20 mmole), and PyBOP (0.502 g, 0.96 mmole). After stirring for 24 h atrt, the reaction mixture was diluted with brine (20 mL) and extractedwith EtOAc (50 mL). The organic layer was washed with 0.1 M NaOH, 0.1 MHCl, and brine, and dried over MgSO₄. The organic solvents were removedin vacuo to give 1,6-bis[4-(5-amidopentyloxypropyl)-phenoxy]hexane as apale yellow oily residue (0.45 g).

Step 5

A solution of 1,6-bis[4-(5-amidopentyloxypropyl)-phenoxy]hexane (0.45 g,0.4 mmole) obtained in Step 4 above, in anhydrous THF (10 mL) was addedto a solution of LiAlH₄ (0.16 g, 4.22 mmole) in anhydrous THF (40 mL) at0° C. The reaction mixture was stirred for 4 h at 80° C. under nitrogenatmosphere and then quenched by with 10% NaOH (1 mL) at 0° C. After 30min., the reaction mixture was filtered and the precipitate was washedwith 10% MeOH in THF (50 mL). The filtrates were combined and evaporatedin vacuo to give a pale yellow oily residue. Purification by flashsilica column chromatography: 5% MeOH/CH₂Cl₂ to 3% i-PrNH₂ in 10%MeOH/CH₂Cl₂ gave the 1,6-bis[4-(6-aminohexyloxypropyl)-phenoxy]hexane.¹H-NMR (CDCl₃, 299.96 MHz): δ (ppm) 7.40–7.25 (m, 12H), 7.22–7.18 (d,2H), 7.09–7.02 (d, 4H), 6.91–6.88 (d, 2H), 6.81–6.75 (d, 4H), 5.01 (s,4H), 4.8–4.75 (m, 2H), 4.70 (s, 4H), 3.96–3.83 (q, 4H), 3.42–3.34 (m,8H), 2.84–2.64 (m, 8H), 2.62–2.56 (t, 4H), 1.84–1.75 (m, 8H), 1.57–1.50(m, 10H), 1.34–1.23 (m, 10H).

Step 6

A solution of 1,6-bis[4-(6-aminohexyloxypropyl)-phenoxy]hexane (0.16 g,0.15 mmole) obtained in Step 5 above, in EtOH (40 mL) was hydrogenatedunder H₂ (1 atm) atmosphere with 10% Pd/C catalyst (100 mg) at rt for 24h. The catalyst was filtered and the filtrate was concentrated to affordcrude product as a pale yellow oil. Purification by reversed phase HPLC:10 to 50% MeCN/H₂O over 40 min; 20 mL/min; 254 nm provides1,6-bis{4-(N-[2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-hydroxyethyl]aminohexyloxypropyl]-phenoxy}hexane40. H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.35 (d, 2H), 7.18–7.15 (dd,2H), 7.08–7.05 (d, 4H), 6.82–6.77 (m, 6H), 4.65 (s, 4H), 3.96–3.92 (t,4H), 3.45–3.34 (m, 8H), 3.12–3.01 (m, 6H), 2.94–2.89 (t, 2H), 2.62–2.57(t, 4H), 1.86–1.43 (m, 28H); ESMS (C₅₄H₈₀N₂O₁₀): calcd. 917.1, obsd.917.5 [M]⁺, 940.8 [M+Na]⁺.

Example 6 Synthesis of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-(R)-hydroxy-ethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]phenylFollowing FIG. 10

Step 1

A mixture of 4-(N-Boc-2-aminoethyl)aniline 28 (10 g, 42.34 mmole),benzaldehyde (4.52 mL, 44.47 mmole), and molecular sieves 4A (10 g) intoluene (100 mL) was refluxed at 95° C. for 15 h. The reaction mixturewas filtered and the filtrate was concentrated in vacuo to give acolorless oil. The oil was dissolved in MeOH (150 mL) and AcOH (0.5 mL)and NaCNBH₃ (2.79 g, 44.4 mmole) were added. The reaction mixture wasstirred at 0° C. for 1 h and at rt for 2 h and then concentrated invacuo to give a pale yellow oily residue. Purification by flash silicacolumn chromatography: 1/1 hexane/EtOAc gaveN-benzyl-4-(N-Boc-2-aminoethyl)aniline 41 as colorless oil (11.5 g,83%). R_(f)=0.75 in 1/1 hexane/EtOAc. H¹-NMR (CD₃OD, 299.96 MHz): δ(ppm) 7.38–7.2 (m, 5H), 6.87–6.84 (d, 2H), 6.58–6.55 (d, 2H), 4.27 (s,2H), 3.2–3.15 (m, 2H), 2.6–2.56 (t, 2H), 1.41 (s, 9H); ESMS(C₂₀H₂₆N₂O₂): calcd. 326.4, obsd. 328 [M+H]⁺.

Step 2

A mixture of N-benzyl-4-(N-Boc-2-aminoethyl)aniline 41 (10 g, 30.7mmole) and (R)-styreneoxide (3.51 mL, 30.7 mmole) in EtOH (100 mL) wasrefluxed for 48 h. A small aliquot of the reaction mixture was taken outfor liquid chromatographic analysis, which indicated that the desiredadduct 2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-1-phenylethanol wasformed as a minor product along with another regio-isomer2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-2-phenyl-ethanol in a ratioof ˜1/2. Evaporation of the solution afforded thick, pale yellow oil,which was purified by flash silica column chromatography: 4/1 to 2/1hexane/EtOAc. After repeated chromatography,2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-1-phenyl-ethanol wasobtained as a colorless oil (4.01 g, 29%) (R_(f)=0.76 in 2/1hexane/EtOAc). H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.4–7.1 (m, 10H),7.1–7.06 (d, 2H), 6.68–6.65 (d, 2H), 5.0 (t, 1H), 4.52–4.46 (d, 1H),4.26–4.22 (d, 1H), 3.76–3.68 (dd, 1H), 3.56–3.48 (dd, 1H), 3.22–3.12 (m,2H), 2.68–2.56 (m, 2H), 1.41 (s, 9H); ESMS (C₂₈H₃₄N₂O₃): calcd. 446.6,obsd. 447.1 [M+H]⁺, 893.4 [2M+H]⁺.

Step 3

To a solution of2-[(N-benzyl-4-[2-N-Boc-aminoethyl)anilino]-1-phenyl-ethanol (4.01 g,8.99 mmole) in CH₂Cl₂ (15 mL) maintained in an ice bath was added TFA(15 mL) under stream of nitrogen atmosphere. After stirring at 0° C. for30 min., the reaction mixture was concentrated in vacuo, yielding a paleyellow oil. Purification by flash silica column chromatography: (½hexane/EtOAc to 5% i-PrNH₂ in ½ hexane/EtOAc) gave2-[(N-benzyl-4-[2-aminoethyl)anilino]-1-phenyl-ethanol 42 as a paleyellow oil from such fractions with R_(f) of 0.2 (5% i-PrNH₂ in ½hexane/EtOAc) in 74% yield (2.29 g). H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm)7.38–7.06 (m, 10H), 7.01–6.98 (d, 2H), 6.71–6.68 (d, 2H), 5.02–4.96 (dd,1H), 4.54–4.48 (d, 1H), 4.29–4.23 (d, 1H), 3.76–3.67 (dd, 1H), 3.58–3.50(dd, 1H), 2.82–2.74 (t, 2H), 2.64–2.59 (t, 2H); ESMS (C₂₃H₂₆N₂O₁):calcd. 346.5, obsd. 346.3[M]⁺,

Step 4

A mixture of 2-[(N-benzyl-4-[2-aminoethyl)anilino]-1-phenylethanol 42(2.28 g, 6.59 mmole), benzaldehyde (0.74 mL, 7.28 mmole), and molecularsieves 4A (4 g) in toluene (40 mL) was heated at 90° C. for 14 h. Thereaction mixture was cooled and filtered, and the sieves were rinsedwith toluene. The combined filtrates were concentrated to give an oilyresidue which was washed with hexane, and dried. The residue wasdissolved in MeOH (40 mL) containing AcOH (0.4 mL) and the reactionmixture was cooled in an ice bath. NaCNBH₃ (0.62 g, 9.87 mmole) wasadded and the reaction mixture was stirred for 2 h at rt, and thenconcentrated. The oily residue was dissolved in 60% MeCN/H₂O, andpurified by reversed phase preparative liquid chromatography (40 to 80%MeCN/H₂O over 30 min; 30 mL/min) to give2-[(N-benzyl-4-[2-N-benzylaminoethyl)anilino]-1-phenylethanol as the TFAsalt. The product was treated with alkaline brine solution, andextracted with ether (200 mL). The organic layer was dried with NaSO₄,and concentrated, to give2-[(N-benzyl-4-[2-N-benzylaminoethyl)anilino]-1-phenylethanol 43 as acolorless oil (1.36 g). H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.36–7.06(m, 15H), 6.98–6.95 (d, 2H), 6.69–6.60 (d, 2H), 5.01–4.96 (t, 1H),4.54–4.47 (d, 1H), 4.29–4.24 (d, 1H), 3.73 (s, 2H), 3.72–3.68 (dd, 1H),3.59–3.54 (dd, 1H), 2.80–2.74 (m, 2H), 2.70–2.64 (m, 2H); ESMS(C₃₀H₃₂N₂O₁): calcd. 436.6, obsd. 437.2 [M+H]⁺.

Step 5

A concentrated solution of2-[(N-benzyl-4-[2-N-benzylaminoethyl)anilino]-1-phenylethanol (1.36 g,3.12 mmole) and compound (s)₄-benzyloxy-3-methoxycarbonylstyreneoxide 44(0.887 g, 3.12 mmole; ˜95% ee) (prepared as described in R. Hett, R.Stare, P. Helquist, Tet. Lett., 35, 9375–9378, (1994)) in toluene (1 mL)was heated at 105° C. for 72 h under nitrogen atmosphere. The reactionmixture was purified by flash silica column chromatography (2/1hexane/EtOAc to 3% MeOH in 1/1 hexane/EtOAc) to give1-{2-[N-benzyl-N-2-(4-benzyloxy-3-methoxycarbonylphenyl)-2-(R)-hydroxy]ethylaminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxy)ethylamino]benzene45. (R_(f)=0.62 in 3% MeOH in 1/1 hexane/EtOAc) was obtained as a paleyellow foam (2.0 g, 89%).

H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.67–7.66 (d, 1H), 7.49–7.42 (m,2H), 7.38–7.0 (m, 20H), 6.88–6.85 (d, 2H), 6.65–6.62 (d, 2H), 5.15 (s,2H), 5.05–4.98 (t, 1H), 4.6–4.54 (t, 1H), 4.53–4.46 (d, 1H), 4.28–4.22(d, 1H), 3.84 (s, 3H), 3.72–3.64 (m, 3H), 3.56–3.46 (dd, 1H), 2.74–2.56(m, 6H); ESMS (C₄₇H₄₈N₂O₅): calcd. 720.9, obsd. 721.4 [M+H]⁺, 743.3[M+Na]⁺.

Step 6

To a suspension of LiAlH₄ (0.211 g, 5.56 mmole) in THF (40 mL) cooledwith ice bath was added1-{2-[N-benzyl-N-2-(4-benzyloxy-3-methoxycarbonylphenyl)-2-(R)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene45 (2.0 g, 2.78 mmole) in THF (10 mL) under nitrogen atmosphere. Thereaction mixture was warmed slowly to rt and the stirring was continuedfor 5 h. The reaction was cooled to 0° C., and 10% NaOH (0.5 mL) wasslowly added. After 30 min., a thick gel formed. The gel was dilutedwith THF (300 mL), filtered, and the solid mass was rinsed with THF (50mL). The filtrates were combined, and concentrated in vacuo, yielding anoily residue. The residue was purified by flash silica columnchromatography (2/1 hexane/EtOAc to 3% MeOH in 1/1 hexane/EtOAc) to give1-{2-[N-benzyl-N-2-(4-benzyloxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzeneas a colorless oil (1.28 g, 67%). H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm)7.4–7.0 (m, 22H), 6.85–6.82 (m, 3H), 6.63–6.60 (d, 2H), 5.02–4.94 (m,3H), 4.66 (s, 2H), 4.59–4.54 (dd, 1H), 4.48–4.4 (d, 1H), 4.24–4.16 (d,1H), 3.76–3.7 (d, 1H), 3.69–3.62 (dd, 1H), 3.58–3.52 (d, 1H), 3.50–3.44(dd, 1H), 2.76–2.54 (m, 6H); ESMS (C₄₆H₄₈N₂O₄): calcd. 692.90, obsd.693.5 [M+H]⁺.

Step 7

A solution of1-{2-[N-benzyl-N-2-(4-benzyloxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl]amino]ethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]-benzene(1.28 g, 1.85 mmole) in EtOH (80 mL) was hydrogenated under H₂ (1 atm)with 10% Pd/C (0.6 g) for 36 h. After filtration and rinsing of thecatalyst with EtOH (50 mL), the filtrates were combined, and evaporatedin vacuo, yielding pale yellow foam which was dissolved in 10% MeCN/H₂O,and purified by reversed phase preparative liquid chromatography (10 to30% MeCN/H₂O (containing 0.3% TFA) over 50 min; 30 mL/min; 254 nm) togive 1-{2-[N-2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-(R)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)-amino]benzeneas the TFA salt (0.6 g, 50%). Optical purity of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene46 which was analyzed with capillary electrophoresis by using a chiralmedium, and estimated to be ˜93%.

H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.42–7.28 (m, 8H), 7.26–7.22 (d,2H), 7.18–7.14 (dd, 1H), 6.80–6.77 (d, 1H), 4.88–4.82 (m, 2H), 4.65 (s,2H), 3.5–3.43 (m, 2H), 3.29–3.26 (m, 2H), 3.19–4.14 (m, 2H), 3.06–3.0(m, 2H); ESMS (C₂₅H₃₀N₂O₄): calcd. 422.5, obsd. 423.1 [M+H]⁺, 445.4[M+Na]⁺.

Example 7 Synthesis of1-{6-[N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-[hydroxyethyl]-amino]hexyloxy}-4-{6-[N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]hexyloxypropyl}benzeneFollowing FIG. 11

Step 1

A solution of 3-(4-hydroxyphenyl)-1-propanol (2.0 g, 13.1 mmole) in DMF(5 mL) was added to a solution of DMF (35 mL) containing NaH (1.31 g,60% in mineral oil) at 0° C. under nitrogen atmosphere. The reactionmixture was slowly warmed to 80° C. After stirring for 1 h at 80° C.,the reaction mixture was cooled to 0° C., and 6-bromohexanenitrile (5.78g, 32.83 mmole) was added. The final mixture was re-heated to 80° C.,and stirred for 24 h. The reaction mixture was quenched with saturatedNaCl solution (200 mL), and the product was extracted with EtOAc (300mL). The organic layer was washed with brine solution, dried withNa₂SO₄, and evaporated to dryness, yielding a pale yellow solid.Purification of the crude product by flash silica column chromatography:4/1 to 1/1 hexane/EtOAc provided6-{3-[4-(5-cyanopentyloxy)phenyl]propoxy}hexanenitrile in 30% yield(1.33 g). R_(f)=0.63 in 1/1 EtOAc/hexane. ¹H-NMR (CDCl₃, 299.96 MHz): δ(ppm) 7.09–7.07 (d, 2H), 6.81–6.78 (d, 2H), 3.96–3.92 (t, 2H), 3.42–3.37(m, 4H), 2.64–2.58 (t, 2H), 2.39–2.32 (m, 4H), 1.87–1.52 (m, 14H).

Step 2

A solution of 6-{3-[4-(5-pentyloxy)phenyl]propoxy}hexanenitrile (1.33 g,3.88 mmole) in THF (10 mL) was added to a solution of LiAlH₄ (0.442 g,11.65 mmole) in THF (50 mL) at 0° C. under nitrogen atmosphere. Thereaction mixture was heated slowly to reflux, and stirred for 2 h. Thereaction mixture was cooled to 0° C., and 10% NaOH solution (5 mL) wasslowly added. After 30 min., the reaction mixture was filtered, and thecollected solids were washed with THF (100 mL). The filtrate wasconcentrated to yield a pale yellow oil which was purified by flashsilica column chromatography: 5% MeOH/CH₂Cl₂ to 3% i-PrNH₂/20%MeOH/CH₂Cl₂ to give 6-{3-[4-(6-aminohexyloxy)-phenyl]propoxy}-hexylamineas a colorless oil (0.5 g, 37%) which was converted to the desiredcompound by proceeding as described in Example 1, step 2 above. Thecrude product was purified by preparatory reversed phase HPLC: 10 to 40%MeCN/H₂O over 40 min; 20 mL/min; 254 nm. ESMS (C₃₉H₅₈N₂O₈): calcd.682.8, obsd. 683.6 [M+H]⁺, 797.5 [M+CF₃CO₂H]⁺.

Example 8 Synthesis ofbis{2-{2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy]ethyamino}-2-hydroxyethoxy}benzeneFollowing FIG. 12

Step 1

To a N₂-saturated solution of acetonitrile (300 mL) containing methyl5-acetylsalicylate 50 (20 g, 0.1 mole) and benzylbromide (13.5 mL, 0.11mole) was added K₂CO₃ (28.5 g, 0.21 mole). The reaction mixture wasstirred at 90° C. for 5 h. After cooling, the reaction mixture wasfiltered, and the filtrate was concentrated, in vacuo, yielding a whitesolid which was susended in hexane (300 mL), and collected on Buchnerfunnel to give methyl O-benzyl-5-acetylsalicylate 51 as colorless towhite crystals (28.1 g, 96%). R_(f)=0.69 in 1/1 EtOAc/hexane. H¹-NMR(CDCl₃, 299.96 MHz): δ (ppm) 7.8.43–8.42 (d, 1H), 8.1–8.04 (dd, 1H),7.5–7.28 (m, 5H), 7.08–7.04 (d, 1H), 5.27 (s, 2H), 3.93 (s, 3H), 2.58(s, 3H).

Step 2

To a solution of methyl O-benzyl-5-acetylsalicylate 51 (14.15 g, 0.05mole) in CHCl₃ (750 mL) was added bromine (2.70 mL, 0.052 mole). Thereaction mixture was stirred at rt. While being stirred, the reactionmixture gradually turned from red-brown to colorless. The mixture wasstirred for 2 h at rt, and quenched by adding brine solution (300 mL).After shaking the mixture in a separatory funnel, organic layer wascollected, washed with brine, and dried under Na₂SO₄. The organicsolution was concentrated in vacuo, yielding white solid. It was washedwith ether (200 mL). After drying in air, 15 g (83%) of methylO-benzyl-5-(bromoacetyl)-salicylate 52 was obtained. R_(f)=0.76 in 1/1EtOAc/hexane. H¹-NMR (CDCl₃, 299.96 MHz): δ (ppm) 8.48–8.46 (d, 1H),8.14–8.08 (dd, 1H), 7.51–7.3 (m, 5H), 7.12–7.09 (d, 1H), 5.29 (s, 2H),4.42 (s, 2H), 3.94 (s, 3H).

Step 3

To a solution of DMF (60 mL) containing methylO-benzyl-5-(bromoacetyl)-salicylate 52 (7.08 g, 0.019 mole) was addedNaN₃ (1.9 g, 0.029 mole). After stirring at rt for 24 h in the dark, themixture was diluted with EtOAc (200 mL), and washed with brine solution(3×200 mL) in a separatory funnel. The organic phase was dried underMgSO₄, and concentrated to afford pale red solid. It was purified byflash silica column chromatography: 10 to 50% EtOAc in hexane. Thedesired product methyl O-benzyl-5-(azidoacetyl)salicylate 53 wasobtained as white crystals (4.7 g, 74%). R_(f)=0.68 in 1/1 EtOAc/hexane.H¹-NMR (CDCl₃, 299.96 MHz): δ (ppm) 8.38–8.36 (d, 1H), 8.08–8.04 (dd,1H), 7.5–7.3 (m, 5H), 7.12–7.09 (d, 1H), 5.29 (s, 2H), 4.53 (s, 2H),3.94 (s, 3H).

Step 4

To a gray suspension of LiAlH₄ (2.74 g, 0.072 mole) in THF (400 mL)cooled in ice bath was added methyl O-benzyl-5-(azidoacetyl)salicylate53 (4.7 g, 0.014 mole) under nitrogen atmosphere. The reaction mixturewas stirred at 0° C. for 1 h, and gradually warmed to rt. After stirringfor 16 h at rt, the mixture was heated at 75° C. for 3 h. The reactionmixture was cooled in ice bath, and quenched by slowly adding 10% NaOH(10 mL). After stirring for 1 h, precipitates were filtered, and rinsedwith 5% MeOH in THF (200 mL). Filtrates were combined, and concentratedin vacuo, yielding pale yellow oily residue. The crude product waspurified by flash silica column chromatography: 10% MeOH/CH₂Cl₂ to 5%i-PrNH₂ in 30% MeOH/CH₂Cl₂ to give2-(4-benzyloxy-3-hydroxymethylphenyl)-2-hydroxyethylanine 39 as a paleyellow solid (2.6 g, 66%). R_(f)=0.63 in 5% i-PrNH₂ in 30% MeOH/CH₂Cl₂.H¹-NMR (CD₃OD, 299.96 MHz): δ (ppm) 7.46–7.28 (m, 6H), 7.24–7.20 (dd,1H), 7.0–6.96 (d, 1H), 5.11 (s, 2H), 4.70 (s, 214), 4.65–4.60 (t, 1H),2.83–2.81 (d, 2H); ESMS (C₁₆H₁₉N₁O₃): calcd. 273.3, obsd. 274.7 [M+H]⁺,547.3 [2M+H]⁺.

Step 5

To a solution of EtOH (15 mL) containing compound2-(4-benzyloxy-3-hydroxymethylphenyl)-2-hydroxyethylamine 39 (0.3 g, 1.1mmole) was added resorcinol diglycidyl ether (0.122 g, 0.55 mmole)dissolved in EtOH (5 mL). The reaction mixture was refluxed for 20 h.After cooling down to rt, the reaction mixture was degassed withnitrogen and hydrogenated with 10% Pd/C (0.3 g, 10%) under H₂ (1 atm)atmosphere for 24 h. After filtration of the catalyst, the filtrate wasconcentrated to dryness, yielding a colorless oily residue which waspurified by preparatory reversed phase HPLC (10 to 50% MeCN/H₂O over 40min; 20 mL/min; 254 nm) to givebis{2-{2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy]-ethyamino}-2-hydroxyethoxy}benzene54. ESMS (C₃₀H₄₀N₂O₁₀): calcd. 588.6, obsd. 589.4 [M+H]⁺, 610.7 [M+Na]⁺.

Example 9 Synthesis of1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxy-ethyl]-amino]ethyl}-4-[N-(2-napth-1-yloxymethyl-2-hydroxyethyl)amino]benzeneFollowing FIG. 13

Step 1

A solution of EtOH (50 mL) containing 4-(N-Boc-2-aminoethyl)aniline 28(0.4 g, 1.69 mmole) and 3-(1-naphthoxy)-1,2-epoxypropane 55 (0.33 g,1.65 mmole) was refluxed for 18 h, and concentrated in vacuo to dryness,yielding a pale yellow oil. It was dissolved in 10 mL of CH₂Cl₂, cooledin ice bath, and treated with TFA (5 mL). After stirring for 2 h at 0°C., the mixture was evaporated, yielding a pale red oil. It wasdissolved in 30% aqueous acetonitrile, and purified by preparatory HPLC:10 to 30% MeCN/H₂O over 30 min; 20 mL/min; 254 nm. The product 56 wasobtained as colorless oil (260 mg; TFA salt). H¹-NMR (CD₃OD, 299.96MHz): δ (ppm) 8.88–8.25 (dd, 1H), 7.82–7.79 (dd, 1H), 7.51–7.42 (m, 3H),7.39–7.38 (d, 1H), 7.33–7.30 (d, 2H), 7.25–7.23 (d, 2H), 6.91–6.89 (d,1H), 4.37–4.31 (m, 1H), 4.22–4.19 (m, 2H), 3.69–3.63 (dd, 1H), 3.67–3.54(dd, 1H), 3.17–3.11 (t, 2H), 2.96–2.91 (t, 2H); ESMS (C₂₁H₂₄N₂O₂):calcd. 336.4, obsd. 337.5 [M+H]⁺, 359.6 [M+Na]⁺, 673.4 [2M+H]⁺.

Step 2

To a solution of compound 56 (0.13 g, 0.023 mmole; TFA salt) in 5 mL ofMeOH was added 1.0 M NaOH (1.0 M, 0.46 mL). After homogeneous mixing,the solution was evaporated to dryness. The residue was dissolved in THF(10 mL), followed by addition of glyoxal 12 (52 mg; 0.023 mmole). Theresulting suspension was stirred for 4 h at ambient temperature undernitrogen atmosphere. After cooling of the resulting solution in icebath, an excess amount of 2M BH₃-Me₂S in THF (3 mL; 6 mmole) was addedto the previous reaction solution. The resulting mixture was slowlywarmed to rt, and refluxed for 4 h under N₂ stream. After cooling of thehot solution, 5 mL of MeOH was added to the cooled mixture to quench thereaction mixture under nitrogen atmosphere. After stirring 30 min at rt,the final solution was evaporated in vacuo, yielding a pale brown solid.It was washed with EtOAc/hexane (1/2; 20 mL), and dried. The crudeproduct was dissolved in 50% MeCN/H₂O containing 0.5% TFA, and purifiedby prep-scale high performance liquid chromatography (HPLC) using alinear gradient (5% to 50% MeCN/H₂O over 50 min, 20 mL/min; detection at254 nM). Fractions with UV absorption were analyzed by LC-MS to locatethe desired product1-{2-[N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]-ethyl}-4-[N-(2-napth-1-yloxymethyl-2-hydroxy-ethyl)amino]benzene57. ESMS (C₃₀H₃₄N₂O₅): calcd. 502.6, obsd. 503.2 [M+H]⁺, 525.6 [M+Na]⁺.

Example 10 Synthesis of1,4,7-tris{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}octane

To a suspension ofα,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone 12 (0.45 g, 1.99mmol) in tetrahydrofuran (15 mL) was added a solution of4-(aminomethyl)-1,8-octadiamine (115 mg, 0.66 mmol) in tetrahydrofuran(5 mL). The resulting suspension was stirred for 12 h at ambienttemperature under nitrogen atmosphere. After cooling of the resultingsolution in ice bath an excess amount of 2 M BH₃-Me₂S in hexane (6 mL,12 mmol) was added. The resulting mixture was slowly warmed to rt, andrefluxed for 6 h under nitrogen atmosphere. After cooling, the reactionmixture was quenched with methanol (5 mL). The resulting solution wasstirred at rt for 30 min., and then concentrated in vacuo to give a palebrown solid. The solid was washed with ethyl acetate:hexane mixture(1:2) and then dried. The crude product was dissolved in 50%acetonitrile/water containing 0.5% TFA and purified by HPLC using alinear gradient (5% to 50% MeCN/H₂O over 50 min., 20 mL/min.; detectionat 254 nM). Fractions with UV absorption was analyzed by LC-MS to locatethe desired product. ESMS (C₃₆H₅₃N₃O₉): Calcd. 671.8; Obsd. 671.7.

Example 11 Synthesis of1-{2-[N-2-(4-amino-3,5-dichlorophenyl)-2-(RS)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenylFollowing FIG. 14

Step 1

A solution of 2-(4-aminophenyl)ethylamine 25 (4.70 mL, 36.7 mmol),benzaldehyde (7.46 mL, 73.4 mmol) and 4A molecular sieves (18 g) intoluene (180 mL) was refluxed for 3 h. The reaction mixture was cooledand filtered. The filtrate was concentrated under reduced pressure toafford compound 58 (95% yield).

Step 2

To a cooled a solution of 58 (2.00 g, 6.40 mmol) in ethanol (150 mL) inice bath was slowly added sodium borohydride (361 mg, 9.50 mmol) under anitrogen atmosphere. The reaction mixture was allowed to stir at 0° C.for 1.5 h, and then warmed slowly to room temperature. The reactionmixture was quenched by slowly adding 50% methanol/TFA (5 mL) and thenthe mixture was concentrated under reduced pressure. The resultantresidue was dissolved in EtOAc, and washed with 0.1 M NaOH. After dryingover Na₂SO₄, the organic phase was concentrated in vacuo, and theresidue was purified by flash silica gel chromatography using ethylacetate/hexanes as eluant to give compound 59(80% yield).

Step 3

To a solution of 59 (1.00 g, 3.20 mmol) in methanol cooled with ice bathwas slowly added di-tert-butyl dicarbonate (0.69 g, 3.2 mmol) in coldmethanol (5 mL). The reaction mixture was allowed to stir at 0° C. for0.5 h, and then warmed gradually to room temperature. After stirring for1 h, the reaction mixture was concentrated under reduced pressure, anddried under high vacuum overnight. The oily residue was purified bysilica gel chromatography using ethyl acetate/hexane (2:1) to affordcompound 60 (80% yield).

Step 4

To a solution of 60 (2.09 g, 5.00 mmol) in methanol (45 mL) was addedphenylglyoxal (2.01 g, 15.0 mmol). The reaction mixture was stirred atroom temperature for 1 h, and followed by slow addition of sodiumcyanoborohydride (1.25 g, 20 mmol). The reaction mixture was stirred atroom temperature overnight. The reaction mixture was concentrated underreduced pressure, and the residue was dissolved in methanol. Afterfiltration, the filtrate was concentrated under reduced pressure. Theresidue was purified by silica gel chromatography using hexane/ethylacetate (9:1) to afford compound 61 (26% yield).

Step 5

To a cooled a solution of 61 (3.19 g, 5.90 mmol) in methylene chloride(12 mL) in an ice bath was added slowly trifluoroacetic acid (12 mL)under stream of nitrogen. After stirring the reaction mixture for 1 h atthe same temperature, the mixture was concentrated under reducedpressure to yield compound 62 as an oily residue. The product was driedin vacuo overnight, and was used in next step without furtherpurification (90% yield).

Step 6

To a mixture of 62 (1.87 g, 3.4 mmol) and2,6-dichloro-4-(bromoacetyl)aniline 63 (1.07 g, 3.8 mmol) in DMF (50 mL)was added potassium carbonate (0.96 g, 6.90 mmol). The reaction mixturewas stirred at room temperature for 0.5 h and then at 35° C. for 1 h.The reaction mixture was cooled to room temperature, and sodiumborohydride (0.16 g, 4.10 mmol) in ethanol (50 mL) was added slowly. Thereaction continued overnight at room temperature and then quenched withaqueous NH₄Cl (sat'd). After concentration of the reaction mixture underreduced pressure, the residue was dissolved in ether, and washed withbrine. After drying over MgSO₄, the organic layer was concentrated, andthe residue was purified by silica gel chromatography by eluting withhexane/ethyl acetate (4:1). The product 64 was obtained in 50% yield.

Step 7

A suspension of 64 (107 mg, 0.17 mmol) and palladium hydroxide (25 mg)in ethanol (2.5 mL) was stirred overnight under hydrogen atmosphere (1atm) at room temperature. The mixture was filtered, and the filtrate wasconcentrated to yield crude product which was purified using silica gelchromatography with 6% methanol/dichloromethane (10% yield) to givecompound 65. ESMS (C₂₄H₂₇Cl₂N₃O₂): calcd: 460.4; obsd: 460 [M+H]⁺, 442[M−H₂O]⁺, 921 [2M+H]⁺.

Example 12 Synthesis of1-{2-[N-2-(4-hydroxy-3-formylaminophenyl)-2-(RS)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenylFollowing FIG. 15

Step 1

1-Benzyloxy-4-bromoacetyl-2-nitrobenzene 66 (3.76 g, 10.8 mmol)[prepared as described in Chem. Bull., 25, 1368–1377, (1977)], was addedto a solution of compound 59 (3.4 g, 10.8 mmol) in dimethylformamide(150 mL) at room temperature. After 28 h, the reaction mixture wasdiluted with ether and washed with a dilute solution of aqueous sodiumchloride. The organic layer was separated and dried over sodium sulfate,filtered and concentrated to give a crude oil. Purification with columnchromatography using hexane:ethyl acetate as the eluent providedcompound 67 (95% yield).

Step 2

To a solution of 67 (4.0 g, 6.80 mmol) in ethanol (500 mL) was slowlyadded sodium borohydride (1 g, 26.50 mmol) in portions over 30 min.,under a nitrogen atmosphere. After 6 h, the reaction mixture wasquenched by slowly adding saturated solution of aqueous ammoniumchloride. The solution was diluted with 1 N sodium hydroxide and ethylacetate and hexanes. The organic layer was separated, dried over sodiumsulfate, filtered and concentrated to give compound 68 as an oil whichwas used in the next step without further purification.

Step 3

To a mixture of 68 (3.6 g, 6.1 mmol) and potassium carbonate (3.0 g, 9.2mmol) in dimethylformamide (100 mL) was added alpha-bromoacetophenone(1.28 g, 6.4 mmol) portionwise. The reaction mixture was heatedovernight at 65° C. An additional portion of alpha-bromoacetophenone(0.25 g, 1.25 mmol) was added and heating was continued. After 18 h, thereaction mixture was cooled to room temperature and ethanol (50 mL) wasadded. Sodium borohydride (1.0 g, 26.5 mmol) was added and stirring wascontinued for 2.5 h. The reaction mixture was concentrated and thenmethanol (25 mL) was added and the excess hydride was quenched with theaddition of a saturated solution of ammonium chloride. The reactionmixture was diluted with ethyl acetate and ether. The organic layer wasseparated, dried over sodium sulfate, filtered, and concentrated. Thecrude oil was purified with column chromatography eluting with ethylacetate/hexanes mixture to give compound 69.

Step 4

To a solution of 69 (0.73 g, 1.0 mmol) in a mixture of methanol (20 mL),6 N hydrochloride acid (1 mL) and water (2 mL) was added iron powder(0.56 g, 10.0 mmol). The reaction mixture was heated at 90° C. for 1.5h. The reaction mixture was cooled to room temperature and allowed tostand overnight. Methanol was added and the brown precipitates andunreacted iron was filtered off. The filtrate was concentrated underreduced pressure, to give 70 as a brown solid which was used in the nextstep without further purification.

Step 5

Compound 70 was dissolved in a premixed solution of acetic anhydride (5mL) and formic acid (3 mL) at room temperature. After 3 h, the reactionmixture was diluted with ethyl acetate and evaporated to dryness. Amethanolic solution of 0.5 M sodium hydroxide was added and the reactionmixture was stirred for 6 h at room temperature. The reaction mixturewas treated with methanolic solution of 1 N hydrochloric acid and thenconcentrated to dryness. The residue was redissolved in methanol andfiltered. The filtrate was concentrated to give compound 71 as a brownresidue which was used in the next step without further purification.

Step 6

Palladium on carbon (10%, 0.5 g) was added to a suspension of 71 inmethanol (120 mL) and dimethylformamide (80 mL). The reaction mixturewas purged with nitrogen gas and stirred overnight under hydrogenatmosphere (1 atm) at room temperature. The mixture was filtered, andthe filtrate was concentrated to yield crude product which was purifiedby HPLC (acetonitril/water/1% TFA gradient) to give crude product whichwas purified with column chromatography using methanol/methylenechloride/1% isopropylamine as the eluent to give desired compound1-{2-[N-2-(4-hydroxy-3-formylaminophenyl)-2-(RS)-hydroxyethyl]-aminoethyl}-4-[N-(2-phenyl-2-(RS)-hydroxyethyl)amino]phenyl72.

FORMULATION EXAMPLES Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Example 2

A tablet Formula is prepared using the ingredients below:

Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient 5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg Starch 45.0 mgMicrocrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as 10%solution in sterile water) Sodium carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1.0 mg Total 120.0 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Example 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient 40.0 mg Starch 109.0mg Magnesium stearate 1.0 mg Total 150.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 150 mg quantities.

Example 6

Suppositories, each containing 25 mg of active ingredient are made asfollows:

Ingredient Amount Active Ingredient 25 mg Saturated fatty acidglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Example 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose aremade as follows:

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodiumcarboxymethyl cellulose (11%) 50.0 mg Microcrystalline cellulose (89%)Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purifiedwater to 5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

Example 8

A formulation may be prepared as follows:

Quantity Ingredient (mg/capsule) Active Ingredient 15.0 mg Starch 407.0mg Magnesium stearate 3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 425.0 mg quantities.

Example 9

A formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1.0 mL

Example 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 1–10 g Emulsifying Wax 30 g LiquidParaffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference in its entirety. Such patches may be constructed forcontinuous, pulsatile, or on demand delivery of pharmaceutical agents.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences, edited by E. W. Martin(Mack Publishing Company, 18th ed., 1990).

BIOLOGICAL EXAMPLES Example 1 β2-Adrenergic Receptor In Vitro FunctionalAssay

The β2-adrenergic receptor functional activity of compounds of theinvention was tested follows.

Cell Seeding and Growth:

Primary bronchial smooth muscle cells from a 21 yr. old male (Clonetics,San Diego, Calif.) were seeded at 50,000 cells/well in 24-well tissueculture plates. The media used was Clonetic's SmBM-2 supplemented withhEGF, Insulin, hFGF, and Fetal Bovine Serum. Cells were grown two daysat 37° C., 5% CO₂ until confluent monolayers were seen.

Agonist Stimulation of Cells

The media was aspirated from each well and replaced with 250 ml freshmedia containing 1 mM IBMX, a phospodiesterase inhibitor (Sigma, StLouis, Mo.). Cells were incubated for 15 minutes at 37° C., and then 250ml of agonist at appropriate concentration was added. Cells were thenincubated for an additional 10 minutes. Media was aspirated and 500 mlcold 70% EtOH was added to cells, and then removed to an empty 96-welldeep-well plate after about 5 minutes. This step was then repeated. Thedeep-well plate was then spun in a speed-vac until all EtOH dried off,leaving dry pellets. cAMP (pmol/well) was quantitated using a cAMP ELISAkit from Stratagene (La Jolla, Calif.). EC₅₀ curves were generated usingthe 4-parameter fit equation:y=(a−d)/(1+(x/c)^(b))+d, where, y=cpm a=total binding c=IC₅₀x=[compound] d=NS binding b=slope

Fix NS binding and allow all other parameters to float.

Example 2 β2-Adrenergic Receptor In Vitro Radioligand Binding Assay

The β1/2-adrenergic receptor binding activity of compounds of theinvention can be tested follows. SF9 cell membranes containing either β1or β2-adrenergic receptor (NEN, Boston, Mass.) were incubated with 0.07nM ¹²⁵I-iodocyanopindolol (NEN, Boston, Mass.) in binding buffercontaining 75 mM Tris-HCl (pH 7.4), 12.5 mM MgCl₂ and 2 mM EDTA andvarying concentrations of test compounds or buffer only (control) in96-well plates. The plates were incubated at room temperature withshaking for 1 hour. The receptor bound radioligand was harvested byfiltration over 96-well GF/B filter plates (Packard, Meriden, Conn.)pre-blocked with 0.3% polyethylenimine and washed twice with 200 μl PBSusing cell harvester. The filters were washed thrice with 200 μl PBSusing cell harvester and then resuspended in 40 μl scintillationcocktail. The filter-bound radioactivity was measured with ascintillation counter and IC₅₀ curves are generated using the standard4-parameter fit equation described above.

The foregoing invention has been described in some detail by way ofillustration and example, for purposes of clarity and understanding. Itwill be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

1. A method of treating a mammal having a disease or conditionassociated with β₂ adrenergic receptor activity, the method comprisingadministering to the mammal, a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a therapeutically effectiveamount of a compound of the formula:

or a pharmaceutically-acceptable salt thereof.
 2. A method of treating amammal having a disease or condition associated with β₂ adrenergicreceptor activity, the method comprising administering to the mammal, apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a therapeutically effective amount of a compound of theformula:

wherein the stereochemistry at *C and **C is (RS) and (RS), (R) and (R),(R) and (S), (S) and (R), or (S) and (S); or a pharmaceuticallyacceptable salt thereof.
 3. The method of claim 2, wherein thestereochemistry at *C is (R) and the stereochemistry at **C is (R). 4.The method of any one of claims 1 to 3 wherein the disease or conditionis selected from asthma, chronic bronchitis, chronic obstructivepulmonary disease, nervous system injury, and premature labor.
 5. Themethod of any one of claims 1 to 3 wherein the disease or condition is arespiratory disease.
 6. The method of claim 5 wherein the respiratorydisease is asthma or chronic obstructive pulmonary disease.
 7. Themethod of any one of claims 1 to 3 further comprising administering atherapeutically effective amount of a steroidal anti-inflammatory agent.8. A method of treating asthma in a mammal, the method comprisingadministering to the mammal, a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a therapeutically effectiveamount of a compound of the formula:

wherein the stereochemistry at *C and **C is (R) and (R), or apharmaceutically acceptable salt thereof.
 9. A method of treating asthmain a mammal, the method comprising administering to the mammal, apharmaceutical composition comprising a pharmaceutically-acceptablecarrier; a therapeutically effective amount of a compound of theformula:

wherein the stereochemistry at *C and **C is (R) and (R), or apharmaceutically acceptable salt thereof; and a therapeuticallyeffective amount of a steroidal anti-inflammatory agent.